Use of plant protein homologue on culture medium

Abstract

To provide a medium supplement that does not accompany with proliferation or an animal product of an adhesion factor serum component or undesired adverse effect of recombinant protein production, a cell-proliferating medium that essentially lacks an animal serum-derived component, and a method for proliferating a cell in the medium to produce a cultured meat.SOLUTION: Provided are a cell culture medium supplement including at least one plant protein homologue of animal serum-derived protein, a cell culture medium including a serum-free base medium and one or more plant-based protein, and a method for proliferating a cell in vitro and a method for producing a cultured meat by using the cell culture medium.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 963,808, filed January 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing

[0002] The ASCII file entitled 108912-675980_SL.txt, created on January 19, 2021, containing 55,930 bytes, which was submitted contemporaneously with the filing of this application, is incorporated herein by reference. The sequence listing hereby submitted is identical to the sequence listing forming a part of this application.

[0003] TECHNICAL FIELD

[0003] The present invention relates generally to cell growth. More specifically, the present invention relates to cell growth media essentially devoid of animal serum-derived components and methods of growing cells in the media, thereby producing cultured meat. [Background technology]

[0004]

[0004] The current world population is over 7 billion and is still growing rapidly. To support the nutritional requirements of this growing population, more and more land is allocated to food production. Natural sources are insufficient to meet the demand. This results in food shortages in some parts of the world. In other parts of the world, the problem is addressed by large-scale production of animals in dense factory farms under harsh conditions. This large-scale production not only causes great suffering to the animals, but also increases arsenic levels in meat products and drug-resistant bacteria due to the organic arsenic compounds and antibiotics used to increase food efficiency and control infections, thus further increasing the number of diseases in both animals and humans and worsening their consequences. Large-scale slaughter is necessary to meet the current food demand, which can result in large-scale disease outbreaks, such as the emergence of porcine pestivirus and mad cow disease. These diseases create a shortage of meat for human consumption, thus completely negating the purpose of animals being raised in the first place.

[0005]

[0005] Furthermore, large scale production reduces the flavour of the final product. It is preferred to be able to supply battery-free eggs and battery-free produced meat. It is not only a matter of taste, but also a health choice, thereby avoiding the consumption of various food additives such as growth hormones. Another problem associated with large amounts of animal production is the environmental problem caused by the enormous amount of waste from the animals, which the environment must subsequently deal with. Furthermore, the large amount of land currently required for the production of animals or animal feed, which cannot be used for alternative purposes such as growing other crops, housing, recreation, wildlife and forests, is a problem.

[0006]

[0006] One of the main problems with the techniques known in the art is that production takes a long time, the cost is very high, and the product is of mediocre quality that cannot and will not replace current meat from livestock. For example, Just-Inc. produced chicken nuggets by growing extracted animal cells in a culture medium, but the production cost is $50 per nugget.

[0007]

[0007] The cultivation of cells, e.g., mammalian or insect cells, for in vitro experiments or ex vivo culture for administration to humans or animals is an important tool in the study and treatment of human diseases. Cell culture is widely used for the production of a variety of biologically active products, e.g., viral vaccines, monoclonal antibodies, polypeptide growth factors, hormones, enzymes, tumor-specific antigens, and food products. However, many of the media used to cultivate cells have limitations. The medium or method contains ingredients that may have a negative effect on cell proliferation and / or maintenance of undifferentiated cell culture. For example, mammalian or insect cell culture media are often supplemented with blood-derived serum, such as fetal calf serum (FCS) or fetal bovine serum (FBS), to provide growth factors, carrier proteins, adhesion and spreading factors, nutrients and trace elements that promote cell proliferation and growth in culture. However, factors found in FCS or FBS, such as transforming growth factor (TGF) beta or retinoic acid, can promote differentiation of certain cell types (Ke et al., Am J Pathol. 137:833-43, 1990) or initiate unintended downstream signaling in cells that promotes undesired cell activity in culture (Veldhoen et al., Nat Immunol. 7(11):1151-6, 2006).

[0008]

[0008] The cost of the culture medium is a major driver of the cost of cultivated meat production. The culture medium is composed of a relatively simple basal medium containing carbohydrates, amino acids, vitamins and minerals, and more expensive serum replacement components including albumin, growth factors, enzymes, attachment factors and hormones. To eliminate the use of animal components, the industry currently relies on recombinant human proteins for applications in cell therapy and vaccine production. However, cultivated meat applications are not limited to the use of human proteins and therefore may be able to utilize more readily available sources of materials suitable for human consumption.

[0009]

[0009] Due to the essentially uncharacterized nature of serum composition and variations between serum lots, the use of serum substitutes and culture in serum-free media are desirable (Pei et al., Arch Androl. 49(5):331-42, 2003). Furthermore, for cells, recombinant proteins or vaccines for therapeutic use grown in cell culture, the addition of animal-derived components is undesirable due to the potential for viral contamination and / or immunogenic effects of animal proteins when administered to humans.

[0010] Serum replacements have been developed to minimize the effects of FCS in cell culture as well as to minimize the amount of animal protein used to culture human cells. Serum replacements, such as KNOCKOUT™ serum replacement (Invitrogen, Carlsbad, Calif.), are culture media of known chemical composition that lack serum and contain basic nutrients and other proteins for cell growth. KNOCKOUT™ cannot be used as a replacement for FBS in plating feeder cells due to the lack of attachment factors, which results in poor cell attachment in this formulation. PC-1™ serum-free medium (Lonza, Walkersville, Md.) is a low-protein serum-free medium formulated in a specially modified DMEM / F12 medium base and contains a complete HEPES buffer system with known amounts of insulin, transferrin, fatty acids, and proprietary proteins.

[0011]

[0011] Cellgro COMPLETE™ (Cellgro, Manassas, Va.) is a serum-free, low-protein culture medium based on a mixture of DMEM / F12, RPMI 1640, and McCoy's 5A basal medium. Cellgro COMPLETE™ does not contain insulin, transferrin, cholesterol, growth or attachment factors, but does contain a mixture of trace elements and high molecular weight carbohydrates, extra vitamins, a non-animal protein source, and bovine serum albumin.

[0012]

[0012] Recombinant proteins produced in animal cells or plants are currently used in culture media. For example, recombinant human albumin is produced in rice, while recombinant fibronectin is produced in mouse cells. There is a need for media supplements that do not carry the animal products of growth or attachment factors serum components or the undesirable side effects of recombinant protein production. The present invention fulfills this long-felt need. Summary of the Invention [Means for solving the problem]

[0013]

[0013] The cost of the culture medium is a major driver of the cost of cultivated meat production. The culture medium is composed of a relatively simple basal medium containing carbohydrates, amino acids, vitamins and minerals, and more expensive serum replacement components including albumin, growth factors, enzymes, attachment factors and hormones. To eliminate the use of animal components, the industry currently relies on recombinant human proteins for cell therapy and vaccine production applications. However, cultivated meat applications are not limited to the use of human proteins and therefore may be able to utilize more readily available sources of materials suitable for human consumption.

[0014]

[0014] The present disclosure is based in part on the discovery that substitutes for some of the most expensive components of serum can be found in protein homologs in the plant kingdom. For example, plant albumins and globulins can surprisingly replace serum albumin as lipid and growth factor carriers in culture media. Catalase is a key enzyme in animal serum that removes hydrogen peroxide and is also abundant in potato, cucumber and other plants. Homologs of common adhesion factors such as fibronectin and vitronectin can be found in plant cell walls and between their membranes. The use of such plant-based proteins in culture media significantly reduces the cost of media for the production of cultivated meat.

[0015]

[0015] One aspect of the present disclosure provides a cell culture medium supplement comprising at least one plant protein homolog of a serum protein.

[0016] In some embodiments, the cell culture media supplement lacks any serum proteins, hi some embodiments, the cell culture media supplement essentially lacks any animal serum-derived components.

[0016]

[0017] In some embodiments, the at least one plant protein homolog comprises a water soluble fraction of a plant protein isolate, hi some embodiments, the water soluble fraction comprises plant albumins and globulins.

[0017]

[0018] In some embodiments, the at least one plant protein homolog is a homolog of serum albumin, serum catalase, serum superoxide dismutase, serum transferrin, serum fibronectin, serum vitronectin, serum insulin, serum hemoglobin, serum aldolase, serum lipase, serum transaminase, serum aminotransferase, serum fetuin, or combinations thereof.

[0018]

[0019] In some embodiments, the at least one plant protein homolog is plant albumin, plant catalase, plant superoxide dismutase, plant transferrin, plant fibronectin, plant vitronectin, plant insulin, plant leghemoglobin, plant aldolase, plant lipase, plant transaminase, plant aminotransferase, plant cystatin, or a combination thereof.

[0019]

[0020] In some embodiments, the supplement comprises plant albumin, plant catalase, plant fibronectin, and plant insulin.

[0021] In some embodiments, the supplement further comprises plant transferrin.

[0020]

[0022] In some embodiments, the supplement further comprises a plant superoxide dismutase.

[0023] In some embodiments, the supplement further comprises plant vitronectin.

[0021]

[0024] In some embodiments, at least one plant protein homolog is a plant albumin. In some embodiments, the plant albumin is chickpea albumin, hemp seed albumin, lentil albumin, pea albumin, soybean albumin, wheat albumin or potato albumin. In some embodiments, the plant albumin is pea albumin or potato albumin.

[0022]

[0025] In some embodiments, the plant albumin is derived from the water soluble fraction of a plant protein isolate.

[0026] In some embodiments, the phytoalbumin has a molecular weight of about 13-110 kilodaltons. In some embodiments, the phytoalbumin has a molecular weight of about 13-17 kilodaltons. In some embodiments, the phytoalbumin has a molecular weight of about 20-35 kilodaltons. In some embodiments, the phytoalbumin has a molecular weight of about 50-110 kilodaltons.

[0023]

[0027] In some embodiments, the phytoalbumin is present in the cell culture medium supplement at a concentration such that the phytoalbumin has a final concentration of about 0.01% to about 10% by weight in the cell culture medium.

[0024]

[0028] In some embodiments, the at least one plant protein homolog is a plant catalase.

[0029] In some embodiments, the plant catalase is Arabidopsis catalase, cabbage catalase, cucumber catalase, cotton catalase, potato catalase, pumpkin catalase, spinach catalase, sunflower catalase, tobacco catalase, or tomato catalase. In some embodiments, the plant catalase is cabbage catalase, cucumber catalase, or potato catalase.

[0025]

[0030] In some embodiments, the plant catalase has a molecular weight of about 50-70 kilodaltons.

[0031] In some embodiments, the plant catalase is present in the cell culture media supplement at a concentration such that when the cell culture media supplement is added to the cell culture media, the plant catalase has a final concentration of about 1 ng / ml to about 100 ng / ml in the cell culture media.

[0026]

[0032] In some embodiments, the at least one plant protein homolog is a plant fibronectin.

[0033] In some embodiments, the plant fibronectin is bean fibronectin, chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin, tobacco fibronectin, or wheat fibronectin.In some embodiments, the plant fibronectin is chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin, or wheat fibronectin.

[0027]

[0034] In one embodiment, the plant fibronectin has a molecular weight of about 40 to 60 kilodaltons.

[0035] In some embodiments, the plant fibronectin has a final concentration of about 0.1 μg / ml to about 100 μg / ml in the cell culture medium.

[0028]

[0036] In some embodiments, the at least one plant protein homolog is a plant insulin.

[0037] In some embodiments, the plant insulin is glucokinin, charantin, or corosolic acid.

[0029]

[0038] In some embodiments, the plant insulin has a final concentration of about 0.05 μg / ml to about 10 μg / ml in the cell culture medium.

[0039] In some embodiments, the at least one plant protein homolog is plant transferrin.

[0030]

[0040] In some embodiments, the at least one plant protein homolog is a plant vitronectin.

[0041] In some embodiments, the at least one plant protein homolog is a plant superoxide dismutase.

[0031]

[0042] In some embodiments, the at least one plant protein homologue is in the form of a plant extract fraction or in a pure form.

[0043] Yet another aspect of the present disclosure provides a cell culture medium comprising a serum-free medium and any of the cell culture medium supplements disclosed herein.

[0032]

[0044] In some embodiments, the cell culture medium lacks any animal serum proteins.

[0045] In some embodiments, the cell culture medium is essentially devoid of any animal serum-derived components.

[0033]

[0046] In some embodiments, the serum-free medium is a basal medium. In some embodiments, the basal medium is a base physiological buffer.

[0034]

[0047] Yet another aspect of the present disclosure provides a kit comprising any of the cell culture media supplements disclosed herein and instructions for mixing the supplement with any serum-free medium lacking animal components and / or animal proteins.

[0035]

[0048] Yet another aspect of the disclosure provides methods of producing cultured meat and producing meat from cultured cells by culturing cells in any of the cell culture media disclosed herein.

[0036]

[0049] In some embodiments, the cells are from a food animal, hi some embodiments, the food animal is a livestock animal, a game animal, a poultry animal, a fish, or a crustacean.

[0050] In some embodiments, the method includes culturing cells, and the cells are fibroblasts, hi one embodiment, the fibroblasts are bovine fibroblasts or chicken fibroblasts.

[0037]

[0051] Yet another aspect of the disclosure provides cultured meat produced by the methods described above and disclosed herein.

[0052] Yet another aspect of the present disclosure provides a method for producing a cell culture medium lacking any animal proteins and / or animal components, the method comprising mixing a serum-free basal medium essentially lacking any animal serum-derived components with any of the cell culture medium supplements disclosed herein essentially lacking any animal serum-derived components.

[0038]

[0053] Yet another aspect of the present disclosure provides a cell culture medium produced by the above method.

[0054] A further aspect of the present disclosure provides the use of plant protein homologues of animal protein in place of animal protein in cell culture medium supplement.In one embodiment, the animal protein is serum protein.In some embodiments, the supplement is essentially devoid of any animal serum-derived components.

[0039]

[0055] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0040]

[0056] The foregoing aspects and other features of the present invention are explained in the following description with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0041] [Figure 1]

[0057] FIG. 1 shows an alignment of albumin homologues of legumes to serum albumin (SEQ ID NOs: 1 to 13). [Figure 2-1]

[0058] FIG. 2 is a diagram showing an alignment of serum albumin and seed storage albumin homologues (SEQ ID NOs: 14 to 43). [Figure 2-2] FIG. 2 is a diagram showing an alignment of serum albumin and seed storage albumin homologues (SEQ ID NOs: 14 to 43). [Figure 3A]

[0059] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 3B] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 3C] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 3D] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 3E] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 3F] Figures 3A-3E show mass spectrometry (MS) analyses of extracted potatoes, and Figure 3F shows SDS-PAGE analyses of extracted potatoes. [Figure 4]

[0060] FIG. 4 shows an SDS-PAGE analysis of pea proteins. [Diagram 5]

[0061] FIG. 5 shows SDS-PAGE analysis of the water-soluble protein fractions of five plant powders (durum, chickpea, lentil, corn, rice) and two commercial plant protein isolates (hemp, pea). [Figure 6]

[0062] FIG. 6 shows the results of MS analysis of four potato extracts from two potato types (red or white). [Figure 7]

[0063] FIG. 7 shows adhesion of soybean, chickpea, lentil, rice and wheat extracts in cultured cells in the absence of serum and animal-derived ECM proteins. [Figure 8]

[0064] FIG. 8 is a schematic diagram showing the preparation of a complete protein bulk as an alternative to bovine serum albumin (BSA). [Figure 9]

[0065] FIG. 9 shows SDS-PAGE analysis of soybean protein (water-soluble fraction) before and after Albusorb purification. [Figure 10A]

[0066] Figure 10A shows MS analysis of the top 10 water-soluble soybean proteins and Figure 10B shows MS analysis of the top 10 Albusorb purified soybean proteins. [Figure 10B] Figure 10A shows MS analysis of the top 10 water-soluble soybean proteins and Figure 10B shows MS analysis of the top 10 Albusorb purified soybean proteins. [Figure 11]

[0067] FIG. 11 shows mass spectrometry (MS) analysis of chickpea proteins. [Figure 12]

[0068] FIG. 12 shows the effect of different plant water soluble fraction proteins on chicken fibroblast cells using a special serum-free supplement lacking BSA protein. [Figure 13]

[0069] FIG. 13 shows the dose-dependent effect of both chickpea and organic pea protein on chicken fibroblasts in suspension culture replacing BSA in serum-free medium. [Figure 14]

[0070] FIG. 14 shows dose-dependent toxicity of chickpea proteins. [Figure 15]

[0071] FIG. 15. Chickpea protein optimization for cell growth. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042]

[0072] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to embodiments and specific language will be used to describe the same, but no limitation of the scope of the disclosure is intended thereby, and it is believed that such modifications and further alterations of the disclosure as set forth herein would normally occur to one of ordinary skill in the art to which the disclosure pertains.

[0043]

[0073] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0074] As used herein, the term "animal component" or "animal components" refers to a composition in which the components are derived, obtained, produced, or produced from an animal. Components are not "animal components" if they are recombinantly produced or derived from a source other than a plant or animal. As used herein, "animal component" does not include recombinant production of components of media in a cell line, including recombinant animal components. Also, "animal component" does not include components produced in an animal cell line.

[0044]

[0075] As used herein, the term "animal serum derived component" or "animal serum derived components" refers to compositions in which components are derived, obtained, produced, or produced from animal serum. Components are not "animal serum derived components" if they are recombinantly produced or derived from sources other than plants or animal serum. As used herein, "animal serum derived components" does not include recombinant production of components of media in cell lines, including recombinant animal components. Also, "animal serum derived components" does not include components produced in animal cell lines.

[0045]

[0076] As used herein, "devoid of" or "free" (as in "free from animal components"), "essentially devoid of" or "essentially free" means non-detectable or small or trace amounts of a component. The term "non-detectable" is understood to be based on standard methodologies of detection known in the art at the time of this application. In some embodiments, "small amount" refers to less than 1% by weight.

[0046]

[0077] As used herein, "animal component-free," "devoid of animal components," or "essentially devoid of animal components" refers to a composition in which the components are not derived, obtained, produced, or produced from an animal. The components may be either recombinantly produced or derived from a source other than a plant or animal. As used herein, "animal component-free," "devoid of animal components," or "essentially devoid of animal components" refers to a composition in which the components are not derived, obtained, produced, or produced from an animal. This allows for the recombinant production of the components of the medium in a cell line of the same origin.

[0047]

[0078] As used herein, the terms "basal media", "basal medium", "base media", "base medium", "base nutritive medium", or "base nutrient media" refer to a basal salt nutrient(s) or an aqueous solution of salts and other elements that provide cells with water and certain bulk inorganic ions essential for normal cellular metabolism, as well as maintaining intracellular and extracellular osmotic balance. In some embodiments, the basal medium includes at least one carbohydrate as an energy source, and / or a buffer system that maintains the medium within a physiological pH range. Examples of commercially available basal media include, but are not limited to, phosphate buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's medium (BME), RPMI 1640, Ham's F-10, Ham's F-12, alpha-Minimum Essential Medium (alphaMEM), Glasgow Minimum Essential Medium (G-MEM), Iscove's modified Dulbecco's medium, or multipurpose medium modified for use with pluripotent cells such as X-VIVO (Lonza), or hematopoietic basal medium.

[0048]

[0079] As used herein, "B27 supplement" (also known as "B22 supplement") is a media supplement containing, for example, 21 components assembled in Neurobasal medium (Invitrogen 21103-049 x 2 units) and 100 g of BSA (e.g., Fraction V IgG Fatty Acid Free Low Invitrogen 30036578 x 1 unit). The following 21 ingredients are present in B27 supplement: 1) Catalase, 2) Reduced Glutathione, 3) Human Insulin, 4) Superoxide Dismutase (SOD), 5) Human Holo-Transferrin, 6) T3, 7) L-Carnitine, 8) Ethanolamine, 9) D+-Galactose, 10) Putrescine, 11) Sodium Selenite, 12) Corticosterone, 13) Linoleic Acid, 14) Linolenic Acid, 15) Progesterone, 16) Retinol Acetate, 17) DL-Alpha Tocopherol (vit E), 18) DL-Alpha Tocopherol Acetate, 19) Oleic Acid, 20) Pipecolic Acid, and 21) Biotin. B27 supplements are modified as follows: Vitamin A-free B27 supplement: retinol acetate is removed, T3-free B27 supplement: T3 (#6) is omitted where appropriate, Antioxidant (AO)-free B27 supplement: the following five antioxidants should be omitted: #1, #2, #4, #17, #18, and BSA-free B27 supplement: BSA is removed. If necessary, human recombinant albumin or serum albumin can be used instead.

[0049]

[0080] As used herein, "complete medium" refers to a basal medium that further contains added supplements that can contribute to cell growth, such as growth factors, hormones, proteins, serum or serum replacements, trace elements, sugars, antibiotics, antioxidants, etc. For example, commercially available complete media contain supplements such as ethanolamine, glutathione (reduced), ascorbic acid phosphate, insulin, human transferrin, lipid-rich bovine serum albumin, trace salts, sodium selenite, ammonium matavanadate, copper sulfate, and manganese chloride (DMEM ADVANCED™ medium, Life Technologies).

[0050]

[0081] As used herein, the term "connective tissue cells" refers to the various cell types that make up connective tissue. For example, connective tissue cells are fibroblasts, chondrocytes, bone cells, adipocytes and smooth muscle cells, or cell types that can naturally differentiate from fibroblasts. As used herein, the terms "natural differentiation" or "naturally differentiated forms" are used to refer to differentiation that occurs in nature, as opposed to one that can be artificially achieved in a laboratory. It is not used to refer to spontaneous transdifferentiation, nor is it used to refer to dedifferentiation. Cell types that can spontaneously differentiate from fibroblasts include chondrocytes, adipocytes, osteoblasts, osteocytes, myofibroblasts, myoblasts, and muscle cells. Connective tissue cells are not mesenchymal stem cells (MSCs) or MSC-derived cells or pluripotent cells.

[0051]

[0082] As used herein, the phrase "spontaneously immortalized fibroblast" refers to a fibroblast that is capable of unlimited cell differentiation, and preferably also cell proliferation, without being subjected to artificial mutations, e.g., genetic engineering, that result in immortalization. Spontaneously immortalized fibroblasts are non-genetically modified.

[0052]

[0083] As used herein, "liquid base mixture" or "base physiological buffer mixture" refers to a base solution of serum replacement or medium supplement in which liposomes are suspended to complete a cell culture medium composition. It is contemplated that the liquid base mixture is loaded into liposomes such that when fused / taken up by cells in cell culture, the liposomes deliver the liquid base mixture to the cells. It is also contemplated herein that the liquid base mixture or base physiological buffer mixture is a basal medium, a complete medium or a physiological buffer solution, such as phosphate buffered saline (PBS) and other balanced salt solutions, that can be used in conjunction with liposomes and / or other components herein to form a serum replacement, a complete medium, a medium supplement, or a cell cryopreservation medium.

[0053]

[0084] As used herein, "culture medium" or "cell culture medium" refers to a water-based solution that provides for the growth, viability, or preservation of cells. Media contemplated herein can be supplemented with one or more nutrients to promote a desired cellular activity, such as cell viability, growth, proliferation, or differentiation of cells cultured in the medium. Media, as used herein, includes serum replacements, media supplements, complete media, or cell cryopreservation media. The pH of the culture medium should be suitable for the microorganisms to grow. Most bacteria grow at pH 6.5-7.0, while most animal cells grow at pH 7.2-7.4.

[0054]

[0085] As used herein, "medium supplement" refers to an agent or composition that is added to a basal medium prior to culturing cells. A medium supplement can be an agent beneficial to cell growth in culture, such as growth factor(s), hormone(s), protein(s), serum or serum replacement, trace element(s), sugar(s), antibiotic(s), antioxidant(s), etc. Typically, a medium supplement is a concentrated solution of the desired supplement that is diluted into a complete or basal medium to reach a final concentration appropriate for cell culture.

[0055]

[0086] As used herein, "serum replacement" or "serum replacement medium" refers to a composition that can be used in conjunction with a basal medium or as a complete medium to promote cell growth and survival in culture. Serum replacements are used in basal or complete media as a replacement for any serum that is characteristically added to media for the culture of cells in vitro. It is contemplated that serum replacements include proteins and other factors for the growth and survival of cells in culture. The serum replacement is added to the basal medium prior to use in cell culture. It is further contemplated that the serum replacement can include basal medium and basic nutrients such as salts, amino acids, vitamins, trace elements, antioxidants, etc., such that the serum replacement is useful as a serum-free complete medium for cell culture.

[0056]

[0087] In this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have their meanings according to U.S. patent law; for example, they may mean "includes," "included," "including," and the like. and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, e.g., allowing for elements not expressly recited, but excluding elements found in the prior art or which affect a basic or novel characteristic of the invention.

[0057]

[0088] Disclosed herein are cell culture media supplements comprising at least one plant protein homologue of an animal protein and methods of making the same. In some embodiments, the animal protein is a serum protein. Also disclosed herein are methods of culturing cells in the disclosed cell culture media supplements and utilizing the cultures for the production of cultivated meat. The cell culture media supplements disclosed herein may also be used in cell culture media and kits. Surprisingly, it has been discovered that plant protein homologues may be utilized in cell culture in a manner that allows cell culture to be devoid of any animal protein and / or animal components. The use of such plant-based proteins in culture media significantly reduces the cost of media for the production of cultivated meat.

[0058]

[0089] One aspect of the present disclosure provides a cell culture medium supplement comprising at least one plant protein homologue of an animal protein. The animal protein can be a serum protein. Thus, in some embodiments, a cell culture medium supplement is provided comprising at least one plant protein homologue of a serum protein. The supplement can be devoid of any animal serum protein. The supplement can be essentially devoid of any animal serum-derived components.

[0059]

[0090] The at least one plant protein homolog can be a homolog of serum albumin, serum catalase, serum superoxide dismutase, serum transferrin, serum fibronectin, serum vitronectin, serum insulin, serum hemoglobin, serum aldolase, serum lipase, serum transaminase, serum aminotransferase, serum fetuin, or combinations thereof.

[0060]

[0091] In some embodiments, the at least one plant protein homolog is plant albumin, plant catalase, plant superoxide dismutase, plant transferrin, plant fibronectin, plant vitronectin, plant insulin, plant leghemoglobin, plant aldolase, plant lipase, plant transaminase, plant aminotransferase, plant cystatin, or a combination thereof.

[0061]

[0092] In some embodiments, the at least one plant protein homologue comprises a plant albumin, a plant catalase, a plant fibronectin, and a plant insulin. In some embodiments, the at least one plant protein homologue further comprises a plant vitronectin. In some embodiments, the at least one plant protein homologue further comprises a plant superoxide dismutase. In some embodiments, the at least one plant protein homologue further comprises a plant transferrin.

[0062]

[0093] At least one plant protein homologue may be a plant albumin homologue. In some embodiments, the plant albumin homologue is derived from a water-soluble fraction of a plant protein isolate. The water-soluble fraction of a plant protein isolate may include plant albumins and globulins.

[0063]

[0094] Albumins are a family of globular proteins, usually related to the globulin protein family. Albumins are water-soluble proteins, moderately soluble in concentrated salt solutions, and undergo thermal denaturation.

[0064]

[0095] Animal albumins are generally found in plasma. Unlike other blood proteins, they are not glycosylated. The most commonly characterized and medically used albumin is bovine serum albumin (BSA), 65-70 kilodaltons (Kd). These serum albumins contain three homologous domains that assemble to form heart-type proteins. Each domain is the product of two subdomains with a common structural motif. Other albumin types include the storage protein ovalbumin in egg white, and the different storage albumins in the seeds of some plants. Albumins bind to the cell surface receptor albondin, but can also enter the cell membrane by pinocytosis.

[0065]

[0096] Serum albumin plays an important role in maintaining the oncotic pressure of blood and serves as an important carrier protein for delivering fatty acids, lipids, and growth factors to cells. In a preferred embodiment, the albumin homologue is a lipid carrier that can be used at a concentration sufficient to bind at least 50 μM oleic acid.

[0066]

[0097] Plant albumins are abundant in the seeds of many plants, such as pea (Croy et al., Biochem J. 1984 Mar 15;218(3):795-803), lentil (Neves et al., Arch Latinoam Nutr. 1996 Sep;46(3):238-42), and hemp (Wang et al., 2019, Comprehensive Reviews in Food Science and Food Safety, 18(4):936-952). They are also common in the roots of starchy plants such as potato (Jirgensons, 1946, Journal of Polymer Science, 1(6):484-494). Plant albumins usually function as storage proteins. They are usually identified as homodimers of 45-55 Kd (Croy et al., Biochem J. 1984 Mar 15;218(3):795-803).

[0067]

[0098] Plant storage albumins are broken down during seed germination to provide nitrogen and sulfur for seedling development. During seed maturation, these proteins undergo post-translational modification and transport before they are deposited in the vacuole in large quantities and with good stability (Mylne et al., 2014, Functional Plant Biology, 41(7):671-677). Sharma et al. (Planta. 2015 May; 241(5):1061-73)) provide the crystal structure of a plant albumin from chickpea (Cicer arietinum) with a hemopexin fold and hemagglutinating activity. Dziuba et al. (Acta Sci. Pol., Technol. Aliment. 2014, 13(2):181-190) provide a proteomic analysis of albumin and globulin fractions of pea (Pisum Sativum L.) seeds.

[0068]

[0099] Albumin has been identified and purified in potato, pea, corn, soybean, wheat, barley, rye, oats, and millet, replacing bovine serum albumin in culture. In some embodiments, the plant albumin is chickpea albumin, hemp seed albumin, lentil albumin, pea albumin, soybean albumin, wheat albumin, potato albumin, or a combination thereof. In another embodiment, the plant albumin is pea albumin, potato albumin, or a combination thereof. In one embodiment, the plant albumin is pea albumin. In one embodiment, the plant albumin is potato albumin.

[0069]

[0100] In some embodiments, the plant albumin comprises a legume albumin. A non-exhaustive list of examples includes pea (Pisum sativum) (Garden p ea) (UniProtKB: P62931, P62930, P62927, P62926, P62928, P62929), Medicago truncatula (Barrel medic) (UniProtKB: G7KHS2, I3SW97, I3S2Y7, A0A072V8Z6, A0A072UYJ7), Trifolium medium (UniProtKB: A0A392M2G9) and chickpea (Cicer arietinum) (UniProtKB: A0A1S2Z3C2). Alignments of such proteins show substantial sequence variation within the family of proteins that demonstrate high sequence similarity and function (e.g., nutritional preserving activity). For reference, an alignment of the aforementioned proteins is shown in FIG. 1, which shows a comparison of 13 legume albumins that are homologs of serum albumin. In particular, over the sequence of P62927 from 71 to 113 compared to 78 to 122 of A2U01_0001997 (zigzag clover (Trifolium medium)), the alignment of P62927 (legume albumin) with A2U01_0001997 shows that 29 of 43 amino acids are identical, and 34 of 43 amino acids are identical or conserved. In non-conserved regions, the homologs of the present invention have more variation, for example, at least 95% identity, at least 90% identity, at least 85% identity, at least 80% identity, at least 70% identity, at least 60% identity, or at least 50% identity. The alignment illustrates the identification by one of skill in the art of a family of plant proteins that are homologs of a serum protein. With respect to mutations such as substitutions, insertions and deletions, the alignment illustrates areas of high sequence identity and similarity.

[0070]

[0101] In some embodiments, the plant albumin comprises patatin or a patatin homologue. Patatins comprise a family of glycoproteins and major tuber storage proteins. Patatins are found in potato and plants of the Solanum genus, such as pepper, tobacco, and tomato. Patatins have been shown to have esterase activity, including lipid acyl hydrolase (LAH), and acyltransferase activity. Non-limiting examples of plant albumins have been identified in potato extracts by MS and include patatin (UniProtKB: M1AGX5, Q2MYP6, Q2VBI2, Q2VBJ3, A0A097H149) and patatin-like phospholipase domain-containing protein (PNPLA) (UniProtKB: M1B3W0). Serum protein homologs include, but are not limited to, patatin and patatin fragments comprising the amino acid sequences set forth by the following UniProtKB accession numbers: M1AGX5, P15477, Q2MY51, Q2MY37, Q2MY45, Q2MY36, P11768, Q3YJT2, Q2MY52, P15476, Q2MY42, Q2MY41, P07745, Q8LPW4, Q2MY48, Q2MY40, Q2MY44, P15478, Q42502, Q3YJT3, Q3YJT0, Q2MY56, Q2MY58, Q2MY54, Q2MY4 3, Q2MY50, Q2MY60, Q2MY39, Q2MY38, Q3YJT5, Q2MY59, Q2MY55, Q41487, Q3YJT4, Q3YJS9, Q8LSC1, Q2VBI5, A0A1S3YWX7, A0A1J6HXU4, A0A2G3DDK1, A0A1U8EX11, A0A1U8EML3, A0A2G2W4I9, A0A1U8EMR5, A0A2G3DDL4, M1BFJ1, A0A0V0HSH3, A0A1J6KIW4, O24152, A0A1J6I2H9, A0A1U7XFQ0, and A0A1S4BJQ4. In other embodiments, any patatin fragment of whole patatin may correspond in size and location to a fragment set forth in the following UniProtKB accession numbers and may include an amino acid sequence set forth in the following UniProtKB accession numbers: Q9AUH5, Q2VB18, Q2VBJ4, Q9SB18, D1MI89, Q2VBI5, I6XCX7, Q2MYQ6, Q41475, Q2MYG0, Q2VBI9, Q7DMV4, Q2VBJ3, Q2VBI2, and Q2MYP6.As illustrative examples, the 51 amino acid (aa) homologue described in Q9AUH5 is the fragment of Q2MY48 from aa92 to aa142. The 132 aa fragment described in Q2VB19 is the fragment of Q2MY58 from aa216 to aa387. Similarly, the 132 aa fragment described in Q2VBJ4 is aa216. The 18 aa fragment described in Q9SB18 is a fragment of Q8LPW4 from aa 369 to aa 386. Any fragment of patatin corresponding in approximate size and location to the listed examples includes a serum protein homologue of the invention.

[0071]

[0102] In some embodiments, the plant albumin is a seed storage albumin or albumin. Such proteins may include activities and functions such as nutrient retention, antimicrobial or antifungal, serine-type endopeptidase inhibitors. Non-limiting examples include Arachis hypogaea (Peanut) (UniProtKB: Q6PSU2, Q647G9), Fagopyrum esculentum (Common buckwheat) (Polygonum fagopyrum) (UniProtKB: Q2PS07), Ricinus communis (Castor bean) (UniProtKB: P01089, B3EWN4), Sinapis alba (White mustard) (Brassica hirta) (UniProtKB: P15322), Sinapis arvensis (Charlock mustard) (Brassica kaber) (UniProtKB: P38057), Brassica juncea)(Indian mustard)(Sinapis juncea)(UniProtKB:P80207), bok choy (Brassica rapa subsp.chinensis (Pak-choi) (Brassica chinensis) (UniProtKB:P84529), soybean (Glycine max) (Soybean) (Glycine hispida) (UniProtKB:P19594), hemp (Hemp) (UniProtKB:A0A219D1L6), Bertholletia excelsa (Brazil nut) (UniProtKB:P04403, P0C8Y8), Capparis masaikai (Mabinlang) (UniprotKB:P30233, P80352, P80351, P80353), Oriental sesame (Sesamum indicum) (Sesamum orientale (UniProtKB:Q9XHP1, B3EWE9), Common dandelion (Taraxacum officinale) (Leontodon taraxacum) (UniProtKB:P86783), Rape (Brassica napus) (UniProtKB:P24565, P09893, P17333, P27740, P0C8Y8, P80208), Winter squash (Cucurbita maxima) (Pumpkin) (UniProtKB:Q39649), Common sunflower (Helianthus annuus) (UniProtKB:P23110, P15461), Arabidopsis thaliana)(Mouse-ear cress)(UniProtKB:Q9FH31, P15457, P15460, P15458), Lupinus angustifolius(Narrow-leaved blue lupine)(UniProtKB:F5B8W8, Q99235, F5B8X0, F5B8X1), Japonica rice (Oryza sativa subsp.japonica (Rice) (UniProtKB:P29835), Matteuccia struthiopteris (European ostrich fern) (Osmunda struthiopteris) (UniProtKB:P17718), Cucurbita moschata (Winter crookneck squash) (Cucurbita pepo var. moschata) (UniProtKB:P84576), Passiflora edulis (Passion fruit) (UniProtKB:P84884), and Picea glauca (White spruce) (Pinus glauc. a) (UniProtKB: P26986). Such protein alignments show substantial sequence variation, but within a family of proteins that demonstrate a high degree of sequence similarity and function. For example, an alignment of a selection of the aforementioned proteins is shown in FIG. 2, which shows a comparison of 30 seed storage albumin homologues of serum albumin. The alignment illustrates the identification of a family of plant proteins that are homologues of serum proteins. With respect to mutations such as substitutions, insertions and deletions, the alignment illustrates regions of high sequence identity and similarity.

[0072]

[0103] Plant albumins are generally high molecular weight albumins of about 50 to 110 Kd, about 2 Phytoalbumins are classified into average molecular weight albumins of 0-35 Kd, and low molecular weight albumins of about 13-17 Kd. Phytoalbumins usually function as homodimers. In some embodiments, phytoalbumins have a molecular weight of about 13-110 kilodaltons (Kd). In some embodiments, phytoalbumins have a molecular weight of about 13-17 Kd. In some embodiments, phytoalbumins have a molecular weight of about 20-35 Kd. In some embodiments, phytoalbumins have a molecular weight of about 50-110 Kd.

[0073]

[0104] The plant albumin may have a concentration of about 0.01% to about 10% (w / w) in the medium. In some embodiments, the phytoalbumin may have a concentration of about 0.01% to about 5% (w / w) in the medium. In some embodiments, the phytoalbumin is at a concentration of about 0.01% to about 5% by weight in the cell culture medium. In some embodiments, the phytoalbumin is at a concentration of about 0.01% to about 0.05% by weight, about 0.01% to about 0.05% by weight, about 0.05% to about 0.1% by weight, about 0.1% to about 0.15% by weight, about 0.15% to about 0.2% by weight, about 0.2% to about 0.25% by weight, about 0.25% to about 0.3% by weight, about 0.3% to about 0.35% by weight, about 0.35% to about 0.4% by weight, about 0.4% to about 0.45% by weight, or about 0.45% to about 0.5% by weight.

[0074]

[0105] The amount of a given component in the cell culture media supplement disclosed herein is It will be understood by those skilled in the art that the concentration of phytoalbumin in the final weight or volume of the culture medium can be calculated to provide a desired concentration. For example, when a cell culture medium supplement is added to a cell culture medium, a person skilled in the art can determine the appropriate concentration of phytoalbumin to be included in the cell culture medium supplement so that the final concentration of phytoalbumin in the cell culture medium is as desired (e.g., 0.01% to about 5% w / w in the final cell culture medium).

[0075]

[0106] The at least one plant protein homologue may be a plant catalase.

[0107] Excess hydrogen peroxide is harmful to almost all cellular components and therefore Fast and efficient removal is very important for aerobically living organisms. Cells grow and proliferate in media supplemented with FBS. It has been known for some time that FBS has a protective role in cell culture and its removal greatly reduces cell viability. Lieberman and Ove (J Exp Med. 1958 Nov 1;108(5):631-7) showed that this reduction in viability was rescued using a protein extract from the liver, the enzyme catalase was then identified as its key component. Thus, chemically defined supplements that support serum-free culture of cells, including B27 supplement (akaB22 supplement from Henna lab), contain many catalases.

[0076]

[0108] Catalase in cell culture is prepared from human red blood cells (e.g. Sigma #C3556) or beef liver (e.g., Sigma #C1345). Catalase is most often derived from bacteria such as Micrococcus lysodeikticus (e.g., Sigma #60634) or Corynebacterium glutamicum (e.g., Sigma #02071), or from Aspergillus niger (e.g., Sigma #106661). It may also be produced from fungi such as fungi (e.g., #C3513).

[0077]

[0109] Catalase (CAT) is an antioxidant enzyme common to almost all living organisms. It catalyzes the decomposition of hydrogen peroxide into water and oxygen, thus protecting cells from oxidative damage caused by reactive oxygen species (ROS). Catalase function is evolutionarily conserved from bacteria to humans (Zamocy et al., Antioxid Redox Signal., 2008 September;10(9):1527-1548). The plant catalase family contains three catalases, which are abundantly expressed in leaves and roots (Sharma and Ahmad, Chapter 4-Catalase: A Versatile Antioxidant in Plants Oxidative Damage to Plants Antioxidant Networks and Signaling, 2014, P.131-148). Active catalase can be isolated from plants very easily and its protective function can be tested in tissue cultures using chemically defined supplements in serum-free cultures where bovine liver catalase is replaced with potato / cabbage catalase (Gholamhoseinian et al., 2006, Asian Journal of Plant Sciences, 5(5):827-831) or cucumber catalase (Hu et al., Genetics and Molecular Biology, 39(3):408-415, 2016).

[0078]

[0110] Catalase is a common water-soluble enzyme found in almost all living organisms exposed to oxygen. It is a catalytic enzyme. It catalyzes the decomposition of hydrogen peroxide into water and oxygen. It helps protect cells from oxidative damage caused by reactive oxygen species (ROS). Its turnover rate is one of the fastest known in nature.

[0079]

[0111] Human catalase is composed of four polypeptide chains, each 500 amino acids long. It contains four iron-containing heme groups that allow the enzyme to react with hydrogen peroxide.

[0112] Plant catalases have their optimum temperature and pH ranges depending on their growth conditions. They are most clearly distinguished from other enzymes that can metabolize peroxidasin by not requiring a reducing agent to catalyze the dismutation reaction (Mhamdi et al., 2010, Journal of Experimental Botany, 61(15):4197-4220). These enzymes consist of polypeptides of mass between 50 and 70 Kd organized into tetramers with each monomer carrying a heme prosthetic group (Regelsberger et al., Plant Physiology and Biochemistry, 2002, 40:479-490).

[0080]

[0113] The second type of heme-dependent catalase is found in some fungi and prokaryotes. It is a structurally distinct protein, a bifunctional catalase-peroxidase (Mutsada et al., Biochemical Journal, 1996, 316:251-257; Regelsberger et al., Plant Physiology and Biochemistry, 2002, 40:479-490).

[0081]

[0114] Martins and English (RedoxBiology, 2014, 2:308-313), catalase activity is stimulated by H2O2 in rich culture medium and is required for H2O2 tolerance and adaptation in yeast.

[0082]

[0115] Multiple molecular forms of catalase are known in different plant species, e.g., tobacco (Nicoti ana tobacco)(Havir and McHale,1987,Plant Physiol.84:450-455.), cotton (Ni et al.,1990,Biochim.Biophys.Acta.1049:219-222), Nicotiana Nicotiana plumbaginlfolia (Willekens et al., 1994, FEBS Lett. 352: 79-83.), Arabidopsis thaliana (Zhong et al. al., 1994, Plant Physiol. 104:889-898), loblolly pine (Pinus taeda) (Mullen and Gifford, 1993, Plant Physiol. 103:477-483), sunflower (Eising et al., 1989, Arch. Biochem. Biophys. 278:258-264), pumpkin (Yamaguchi et al., 1986, Eur. J. Biochem. 159:315-322) and tomato (Gianinetti et al., 1993, Physiol. Plant. 89:157-164). The nomenclature of catalase is also based on its isoforms in different plant species. According to the classification proposed by Willekens et al. (1995, EMBO J. 16:4806-4816), class I, class II and class III catalases are specifically expressed in photosynthetic, vascular and reproductive tissues, respectively. The existence of multiple catalase isozymes suggests the structural and functional diversity of catalases in various plant species. cDNAs of various catalases have been isolated and characterized from different plant species in order to understand the genes and their regulatory components (Scandalios, 1992, Current Communications in Cell and Molecular Biology, 2001). (Biology: Molecular Biology of Free Radical Scavenging Systems (5). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Catalase isozymes show developmental stage and organ specificity in plants.

[0083]

[0116] Plant catalase includes Arabidopsis catalase, cabbage catalase, cucumber catalase, The plant catalase may be cucumber catalase, cotton catalase, potato catalase, pumpkin catalase, spinach catalase, sunflower catalase, tobacco catalase, tomato catalase, or a combination thereof. In one embodiment, the plant catalase is cabbage catalase, cucumber catalase, potato catalase, or a combination thereof. In one embodiment, the plant catalase is cucumber catalase. In one embodiment, the plant catalase is potato catalase.

[0084]

[0117] The plant catalase may have a molecular weight of about 50-70 kilodaltons.

[0118] In some embodiments, the concentration of plant catalase in the medium is about 1 to about 100 ng. The final concentration of plant catalase in the medium can be about 1 g / l to about 5 g / l, and preferably about 2.5 g / l, for example about 7 ng / ml, about 11 ng / ml, about 14 ng / ml, about 18 ng / ml, about 21 ng / ml, about 28 ng / ml, about 35 ng / ml, or about 55 ng / ml. In some embodiments, the final concentration of plant catalase in the medium can be about 1 g / l to about 5 g / l, and preferably 2.5 g / l.

[0085]

[0119] The Uniprot database lists potato catalase (UniprotKB The UniProt database contains at least 128 proteins with 90% identity to potato catalase (UniProtKB:M1ALT0) and at least 958 proteins with 50% identity to potato catalase. Similarly, the UniProt database contains at least 23 proteins with 90% identity to Triticum catalase (UniProtKB:Q43206) and at least 958 proteins with 50% identity to wheat catalase. Exemplary sources of plant catalases and catalases from those plants used in accordance with the present invention include, but are not limited to, soybean (UniProtKB:O48561), chickpea (UniProtKB:A0A1S2Y835, Q9ZRU4), summer squash (UniProtKB:P48350), mung bean (UniProtKB:P32290), common bean (UniProtKB:T2DN96, V7AQS4), and Further examples include cotton (UniProtKB: P17598, A0A5D2M8G9, A0A5J5SMB2). Another green plant database source is Phytozome, the Plant Comparative Genomics portal of the Department of Energy's Joint Genome Institute (Heinze, et al., (2002) Plant Catalases. In: Baker A., ​​Graham IA (eds) Plant Peroxisomes. Springer, Dordrecht.).

[0086]

[0120] As described and exemplified herein, plant homologs of serum catalase and Areas of conserved and non-conserved sequences between proteins and between individual members of the family are readily recognized. With respect to mutations such as substitutions, insertions and deletions, alignments are located to areas of high sequence identity and similarity.

[0087]

[0121] Isolation of plant catalases is well known to those skilled in the art and can involve techniques such as liquid fractionation, centrifugation, column chromatography, and the like. Catalase was identified in the present disclosure in potato.

[0088]

[0122] At least one plant protein homologue is identified: plant fibronectin, plant vit. Lonectin, or a combination thereof.

[0123] Cell survival in culture is often dependent on the surface provided for attachment. In vivo, extracellular matrix (ECM) proteins of basement membranes adhere cells to neighboring tissues by binding the integrin family of surface glycoproteins. Laminin, collagen IV and heparan sulfate constitute the basement membrane proteins in adult tissues, while embryonic and regenerative tissues also exhibit fibronectin. Many of the same ECM proteins are animal-derived or recombinantly expressed and support cell adhesion and proliferation in vitro.

[0089]

[0124] In plants, the role of ECM-like proteins in support and scaffolding has also been recognized. Several animal ECM-like proteins have been discovered in plant cell walls over the years (Seymorr et al. 2004, Biotechnology and Genetic Engineering Reviews, 21(1):123-132). Fibronectin-like proteins are involved in cell wall-cell membrane adhesion and have been shown to be abundant under conditions of salt stress / water deficit (Zhu et al., 1993). Pellenc and colleagues (Pellenc et al., 2004, Protein Expression and Purification 34:208-214) were able to isolate a fibronectin-like protein from plants using a protocol similar to that for isolating fibronectin from human plasma.

[0090]

[0125] A tobacco protein immunologically related to human vitronectin is a non-adapted and salt-dependent It is found in the cell walls and membranes of adapted tobacco cells and is abundant in adapted cells (Zhu, JK et al., Plant J. for Cell and Molecular Biology, 30 Apr 1993, 3(5):637-646). An antibody discovered by Sanders specific for a 55 Kd polypeptide in tobacco cells was also able to recognize human Vn (Sanders et al., 1991, Plant Cell, 3:629-635). A monospecific antibody specific for a 59 Kd protein in tobacco microsomal membranes recognizes human fibronectin.

[0091]

[0126] The plant homologue of fibronectin is bean fibronectin (UniProtK B: V7C3U9, V7CSV1, A0A0L9VRR4, A0A1S3UT35, A0A1S3UV51), chickpea fibronectin (UniProtKB: A0A1S2 Z0R0, A0A1S2YDZ6, A0A1S3E9P2, A0A1S3E9K8, A0A1S2YE00), lentil fibronectin, rice fibronectin (UniProtKB:1NXC4, A0A0E0JVM6, J3L9M7, A0A0E0N9Z2), soybean fibronectin (UniProtKB:I1MSQ1, I1KIT9, K7L4U6, A0A0R0IKE0), tobacco filamentous fibronectin (UniProtKB:1NXC4, A0A0E0JVM6, J3L9M7, A0A0E0N9Z2), It may be wheat fibronectin (UniProtKB: A0A1J6IU80, A0A1S4B3E7, A0A1S4AF52, A0A1J6HY83) or wheat fibronectin (UniProtKB: A0A3B6NN97, A0A3B6KF25, A0A3B6QBJ6) or chickpea disease fungus fibronectin (UniProtKB: A0A163LSC5, A0A163GQI0).

[0092]

[0127] Fibronectin and vitronectin-like proteins have been identified in several plants. and crude plant extracts were used to support cell attachment and proliferation in cell culture medium.

[0093]

[0128] In some embodiments, the at least one plant protein homologue is a plant fibronectin. In some embodiments, the plant fibronectin is bean fibronectin, chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin, tobacco fibronectin, wheat fibronectin, or a combination thereof. In some embodiments, the plant fibronectin is chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin, wheat fibronectin, or a combination thereof. In one embodiment, the plant fibronectin is lentil fibronectin. In one embodiment, the plant fibronectin is rice fibronectin. In one embodiment, the plant fibronectin is soybean fibronectin. In one embodiment, the plant fibronectin is wheat fibronectin.

[0094]

[0129] Plant fibronectin may have a molecular weight of about 40-60 Kd. Fibronectin is often composed of two larger subunits of approximately 250 Kd. In some embodiments, plant fibronectins have a molecular weight of approximately 40-60 Kd.

[0095]

[0130] In some embodiments, plant fibronectin is present at about 0.1 μg / ml to about 0.1 μg / ml in the medium. At a concentration of about 100 μg / ml. In some embodiments, the plant fibronectin is at a concentration of about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 15 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, or about 50 μg / ml in the medium.

[0096]

[0131] In some embodiments, the at least one plant protein homologue is expressed in a plant in vitro. It may be a nectin.

[0132] In some embodiments, plant vitronectin is present in a concentration of about 0.1 μg / ml to about In some embodiments, the plant vitronectin is at a concentration of about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 15 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, or about 50 μg / ml in the medium.

[0097]

[0133] The isolation of plant fibronectin and vitronectin is well known to those skilled in the art. This involves volume fractionation, centrifugation, and column exchange, all of which are routine experiments.

[0134] The at least one plant protein homologue can be a plant leghemoglobin. .

[0098]

[0135] In some embodiments, the plant leghemoglobin is present in a concentration of about 1 μg / ml to about 1 The concentration is 0.00μg / ml.

[0136] Leghemoglobin is expressed in legume plants in response to root colonization by nitrogen-fixing bacteria. Leghemoglobin is an oxygen-carrying protein found in nitrogen-fixing root nodules of legumes, produced by Leucaena ketella. Leghemoglobin includes serum protein homologues of the present invention. Leghemoglobin is found in, but not limited to, plants such as Pisum sativum (Garden pea) (UniProtKB: LGB1_PEA, LGB2_PEA, LGB3_PEA, LGB4_PEA, LGB5_PEA, LGB6_PEA), Medicago sativa (Alfalfa) (UniProtKB: LGB1_MEDSA, LGB2_MEDSA, LGB4_MEDSA, Q42928_MEDSA, Q43786_MEDSA), Canavalia lineata (Beach bean) (Dolichos lineatus) (UniProtKB: LGB_CANLI), Chickpea (Cicer arietinum (Chickpea) (Garbanzo) (UniProtKB: A0A1S2YZ78, A0A1S2XXT5, A0A1S2XKV1, A0A1S2XMF3), Glycine MAX (Soybean) (Glycine hispida) (UniProtKB: A0A0R0HW51, Q96428, Q42801), Glycine soja (Wild soybean) (UniProtKB: A0A445IPX6), and Medicago sativa (Alfalfa) (UniProtKB: P28010, 14962, Q42928, Q43786).

[0099]

[0137] The at least one plant protein homologue may be a plant lipase.

[0138] In some embodiments, the plant lipase is present at a concentration of about 1 μg / ml to about 100 μg / ml in the medium. The concentration is / ml.

[0100]

[0139] Further non-limiting examples of serum protein homologues include the following serum proteins: Lipases include those from plants such as pea (garden pea) (UniProtKB: Q01517), wheat (Triticum aestivum) (UniProtKB: A0A1D5UIX7, A0A2X0SGN9, A0A3B6GZV3, A0A3B6GY43), Arabidopsis thaliana (Mouse-ear cress) (UniProtKB: A0A178WBX6), and many others. Methods for preparing plant lipases are well known to those skilled in the art (see, for example, Wagenknecht, A. C. et al., 1958, Journal of Food Science, 23(5):439-445; Barros, M. et al., 2010, Brazilian Journal of Chemical Engineering 27(1):15-29).

[0101]

[0140] The at least one plant protein homologue may be a plant cystatin.

[0141] In some embodiments, the plant cystatin is present at a concentration of about 1 μg / ml to about 100 μg / ml in the medium. Concentration is g / ml.

[0102]

[0142] Fetuin is a blood protein that is manufactured in the liver and secreted into the bloodstream. These include serum albumin. They belong to a group of binding proteins that mediate the transport and availability of a wide range of cargo substances in the bloodstream. Serum albumin is the most abundant protein in the plasma of adult animals, whereas fetuin is more abundant in fetal blood. Fetuin A is the major carrier protein for free fatty acids in the circulation. Fetuin A is a cellular It has been reported to play a role in adhesion and signal transduction and to regulate the proliferation, motility, and invasion of certain cancer cell types. Fetuin belongs to the cystatin superfamily of proteins and evolved from the protein cystatin by gene duplication and exchange of gene segments. In mammals, fetuin A and fetuin B are paralogous plasma proteins of the cystatin superfamily (see, e.g., Karmilin et al., 2019, Sci Rep. 2019;9:546). Many cystatins have been identified as inhibitors of papain-like cysteine ​​proteinases. Fetuin A is not known to be an inhibitor of proteases, whereas fetuin B selectively inhibits certain metalloproteinases. Mammalian cystatins are generally cysteine ​​protease inhibitors and are found in all biological fluids. Mammalian cystatin C is a secreted protein that is internalized by cells (Ekstrom, U. et al., 2008, FEBS Journal 275:4571-4582) and can be used in cell culture applications to inhibit intracellular processes, including inhibition of polio, herpes simplex and coronavirus replication.

[0103]

[0143] The MEROP database identifies cystatin as a member of the I25 family. The cystatin family (denoted I25) comprises cysteine ​​protease inhibitors, including the cystatin classification, which is subdivided into four subfamilies: I25A, I25B, I25C, and unclassified (Rawlings et al., 2014, Nucleic Acids Res. 42:D503-D509; Martinez, M. et al., 2009, Plant Physiol. 2009, Nov;151(3):1531-45). Plant cystatins (classified as phytocystatins) contain an N-terminal alpha helix (present only in plant cystatins) and have been identified primarily from seeds, with some detected in other plant tissues. Multicystatins in potato (Solanum tuberosum) and tomato (Solanum lycopersicum) can be found in the vacuole and in the cytoplasm (Nissen et al., 2009, Plant Cell 21:861-875; Madureira et al, 2006, Environ Exp Bot 55:201-208).

[0104]

[0144] Plant homologues of serum proteins include plant cystatins. The presence of a helix, which is present only in plant cystatins. Non-limiting examples include Vigna unguiculata (cowpea) cystatin (UniProtKB: A0A4D6KLC0, A0A4D6NH52), soybean (Glycine max) cystatin (UniProtKB: A0A4D6KLC0, A0A4D6NH52), and plant cystatin (UniProtKB: A0A4D6KLC0, A0A4D6NH52). max (Soybean) cystatins (UniProtKB: I1K3Q1, P25973, A0A0R4J598, I1MYC1), Hordeum vulgare (Barley) cystatins (UniProtKB: Q9LEI7), Oryza sativa (rice) cystatins (UniProtKB: A2XS65, Q6K309, A0A1S4AF52, A0A1J6HY83), Solanum tuberosum (potato) cystatins (UniProtKB: P37842, M1B0W4, M1C699, M1B0W5, M1BIR8), and Zea mays (Zea mays (maize) cystatin (UniProtKB: P31726, B6SNY0, B6UGN8), Triticum (wheat) cystatin (UniProtKB: Q8W252), Phaseolus vulgaris (kidney bean) cystatin (UniProtKB: V7C6Q5, V7BNT8), Arachis hypogaea (peanut) cystatin (UniProtKB: A0A445AB69, A0A445DKL4, E5BDA5), Helianthus annuus (common sunflower) cystatin (UniProtKB: Q10992, Q109923), or Dictyostelium discoideum (slime mold) cystatin (UniProtKB: Q10992, Q109924). Cystatin (UniProtKB:Q65YR7, Q65 YR8, Q5R1U3) (see green plant database resources Phytozome and UniProt for cystatins).

[0105]

[0145] The at least one plant protein homologue can be a plant aldolase.

[0146] In some embodiments, the plant aldolase is present at a concentration of from about 1 μg / ml to about 100 μg / ml in the medium. Concentration is in μg / ml.

[0106]

[0147] Aldolase is an enzyme that helps break down certain sugars and is found in abundance in muscle tissue. It is released into the blood and is detectable in the blood. An example of a plant homologue is fructose-1,6-bisphosphate aldolase (FBA), a key plant enzyme involved in glycolysis, gluconeogenesis, and the Calvin cycle (Lv et al., 2017, Front Plant Sci. 8:1030). Another source is pea (UniProtKB: Q01517).

[0107]

[0148] Other suitable plant homologues of proteins include transaminases and asparagine Contains acid aminotransferase.

[0149] At least one plant protein homologue may be a plant transaminase. Non-limiting examples of transaminases include those from garden Pea (Pisum sativum) (UniProtKB: P49364, Q9AVH0, O22464) and Triticum (Wheat) (UniProtKB: P84188). In some embodiments, the plant transaminase is at a concentration of about 1 μg / ml to about 100 μg / ml in the medium.

[0108]

[0150] At least one plant protein homologue is a plant aminotransferase. It is possible. Matheron describes the purification and properties of aminotransferase from pea (Matheron et al., Plant Physiol. 1973, 52:63-67). Non-limiting examples of aspartate aminotransaminases include those from soybean (Glycine max) (UniProtKB:I1JUS6) and wheat (Triticum) (UniProtKB:B5B1F8). In some embodiments, the plant aspartate aminotransferase is at a concentration of about 1 μg / ml to about 100 μg / ml in the medium.

[0109]

[0151] Insulin homologs from plant sources may be used as part of the present disclosure in cell culture supplements. It can also be used as a ment.

[0152] In some embodiments, the structural and functional homologue of insulin is administered in cell culture medium. Examples of such insulin homologues include, but are not limited to, glucokinin, charantin, and corosolic acid. Glucokinin is a structural homologue of insulin. Charantin is found in bitter melon (Momordica Corosolic acid is a mixture of two steroid glycosides derived from the Banaba (Lagerstroemia speciose) plant, or bitter lemon. Corosolic acid is a five-ring triterpene acid found in the Banaba (Lagerstroemia speciose) plant, and is usually extracted from banana leaves.

[0110]

[0153] In some embodiments, the plant insulin is glucokinin, charantin, coenzyme Q10, or the like. In one embodiment, the plant insulin is glucokinin. In one embodiment, the plant insulin is charantin. In one embodiment, the plant insulin is corosolic acid.

[0111]

[0154] In some embodiments, the plant insulin has a molecular weight of about 6 Kd. In this embodiment, the plant insulin is at a concentration of about 0.05 μg / ml to about 10 μg / ml in the medium.

[0112]

[0155] At least one plant protein homologue is a plant superoxide dismutase In some embodiments, the plant SOD has a molecular weight of about 80 to 89 Kd. In some embodiments, the plant SOD is at a concentration of about 1 μg / ml to about 20 μg / ml in the medium.

[0113]

[0156] The at least one plant protein homologue can be plant transferrin. .

[0157] The serum replacement proteins specifically identified herein are by way of example and not limitation. It also includes orthologs and / or paralogs of non-animal proteins. Orthologs are often defined as homologous genes or proteins that are the result of a speciation event. In a simple model, after a speciation event, orthologs arise when a gene or protein of a first and second species diverges. Although the sequences of orthologs may differ, orthologous proteins and encoding nucleotides tend to have the same or similar function or activity or to play the same role in different species and are maintained in the speciation event. Paralogs are often defined as homologous genes or proteins that are the result of a duplication event in a species. In a simple model, paralogs arise after a gene duplication event and the divergence of one copy from the other. Paralogs can evolve separately in the same species and therefore tend to be more diverse in their roles, but their functions may be similar. For example, paralogs may have similar enzymatic activity but act on different substrates or be expressed in different tissues or at different developmental stages. The relationship between orthologs and paralogs can be more complex, for example, after gene duplication there is speciation.

[0114]

[0158] Alternative embodiments include serum protein variants that contain mutations, including substitutions, insertions, and deletions. Certain amino acid sequence variants may differ from a reference sequence by the insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, alternative embodiment sequences may include a reference sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues have been inserted, deleted or substituted. For example, 5, 10, 15, up to 20, up to 30 or up to 40 residues may be inserted, deleted or substituted.

[0115]

[0159] For any particular plant homologue of a serum protein, orthologs and paralogs are Comparison of non-animal homologues, including those that are not related to the homologues of the non-animal protein, serves as a guide for the mutations that can be made or selected. For example, it is clear from such sequence alignment which parts of the homologue protein are more or less conserved and which parts may contain more or less variation. Therefore, one way to determine whether a protein is a suitable non-animal serum protein homologue is to align the homologues provided herein, identify which parts of the homologue are more or less conserved or contain more or less variation, or where there may be insertions and deletions, and then compare the protein in question to one or more of the homologues provided herein. Positional alignment algorithms such as Smith and Waterman can be used to align two or more homologues. Such algorithms can be built into computers to optimize the alignment. Several computer programs are available that use the Smith and Waterman algorithm. For example, BestFit uses the Smith-Waterman algorithm to find the best positional alignment between two sequences. Other algorithms may be used, such as BLAST, psiBLAST or TBLASTN (using the method of Altschul Et Al. (1990) J. Mol. Biol. 215:405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448).

[0116]

[0160] By multiple sequence alignment, highly conserved amino acid residues or inversions were identified. Alternatives are identified. Sequence comparison highlights amino acid residues that are identical or nearly identical between sequences and are likely to be important for function, conserved or highly conserved amino acids, and amino acids that are variable. Alignment also shows that there may be insertions or deletions in the protein sequence from one protein to another, and thus not necessarily.

[0117]

[0161] Take any one of the proteins specifically disclosed by reference, and use an ortholog, The homologs of the present invention, including homologs and variants thereof, may differ from the reference in conserved regions by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. A pairwise comparison of the disclosed sequences can be seen as a guide. Conservative substitutions include the substitution of an amino acid with a different amino acid that has similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another non-polar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue, or an aromatic residue may be replaced by another aromatic residue.

[0118]

[0162] Amino acids can be grouped into different classes according to common side chain properties. The amino acids may be grouped into different classes according to their common side chain properties: a. hydrophobic: Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that affect chain orientation: Gly, Pro; aromatic: Trp, Try, Phe. Non-conservative substitutions require the exchange of a member of one of these classes for another. Amino acids may be grouped into different classes according to their common side chain properties: a. hydrophobic: Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that affect chain orientation: Gly, Pro; aromatic: Trp, Try, Phe. Non-conservative substitutions require the exchange of a member of one of these classes for another.

[0119]

[0163] Conservative substitutions as shown in Table 1 below

[0120] [Table 1]

[0121]

[0164] In some embodiments, the identified homologs have at least 50%, at least 55%, %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0122]

[0165] In some embodiments, a homologue has at least 50% sequence identity to the identified homologue. It has sexuality.

[0166] In some embodiments, a homologue has at least 60% sequence identity with the identified homologue. Have identity.

[0123]

[0167] In some embodiments, a homologue has at least 70% sequence identity with the identified homologue. Have identity.

[0168] In some embodiments, a homologue has at least 80% sequence identity with the identified homologue. Have identity.

[0124]

[0169] In some embodiments, a homologue has at least 90% sequence identity with the identified homologue. Have identity.

[0170] In some embodiments, a homologue has at least 95% sequence identity with the identified homologue. Have identity.

[0125]

[0171] Some embodiments include other proteins and polypeptides of serum protein homologs. Fusion proteins may exhibit enhanced production, activity, stability, and / or targeting. Serum protein analogs may be evaluated alone or in combination.

[0126]

[0172] In some embodiments, the plant protein homologue may be glycoengineered. Glycosylation is one of the major post-translational protein modifications. N-linked glycosylation is the attachment of an oligosaccharide, sometimes called a glycan, consisting of several sugar molecules, a carbohydrate, to the nitrogen atom (the amide nitrogen of an asparagine (Asn) residue in a protein). O-linked glycosylation is the attachment of a sugar molecule to the oxygen atom of a serine (Ser) or threonine (Thr) residue in a protein. Glycosylation is often essential for protein structure and function. N- and O-glycans have been shown to play important roles in protein structure, stability, aggregation, and thermal denaturation, and have been observed to affect the pharmacodynamics and pharmacokinetics of recombinant therapeutic proteins. N- and O-linked carbohydrate moieties of plant and insect glycoproteins are also abundant environmental immunodeterminants.

[0127]

[0173] Glycoengineering refers to the selection or remodeling of glycans. Non-human mammalian cells, such as CHO, have been primarily used for the production of biopharmaceuticals with human-like glycosylation profiles.

[0128]

[0174] Yeast and other fungal hosts are important production platforms for the production of recombinant proteins. The glycosylation process is a glycoform that is a glycosylation product of the human genome. A cellular system of the yeast strain Pichia pastoris has been developed to carry out a series of enzymatic reactions that mimic the process of glycosylation in humans. For example, U.S. Patent Nos. 7,029,872, 7,326,681, and 7,449,308 describe methods for producing recombinant glycoproteins similar to human proteins that contain N-linked glycans that are complex sialylated biantennary glycosylation. Glycoengineering can involve expressing a protein in an organism that has been engineered to perform a specific glycosylation.

[0129]

[0175] Glycoengineering involves the engineering of enzymes such as endoglycosidases and glycosynthases. The enzyme may be enzymatic, such as using an enzyme. Exemplary endoglycosidases include, but are not limited to, endo-β-N-acetylglucosaminidase H (Endo-H), a recombinant glycosidase that cleaves within the high mannose chitobiose core and hybrid oligosaccharides of some N-linked glycoproteins. Endo-N-acetylglucosaminidase F2 (Endo-F2) cleaves high mannose and biantennary N-glycans, and endo-N-acetylglucosaminidase F3 (Endo-F3) cleaves triantennary and alpha-(1-6)-fucosylated biantennary N-glycans of peptides and proteins (Plummer et al., Anal Biochem 235:98-101, 1996). Such enzymes can be used to digest oligosaccharides to single sugar units (e.g., GlcNAc), which can then be extended with an oligosaccharide of choice by glycosylation mediated by glycosyltransferases. Alpha-fucosidases can be used to defucosylate terminal asparagine-linked GlcNAc. Glycosyltransferases for extending single sugar units include, but are not limited to, endo-beta-1,4-galactosyltransferases. Oligosaccharides can be sialylated. Sialyltransferases can be used to catalyze the transfer of a sialic acid moiety to the terminal portion of an oligosaccharide acceptor. Each sialyltransferase is specific for a particular sugar substrate.

[0130]

[0176] To completely remove oligosaccharides, the amidase PNGase F is the most It cleaves between the inner GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides, and is effective in removing almost all N-linked oligosaccharides, with additional The N-glycan core oligosaccharide is left intact for analysis (WO 2013 / 120066).

[0131]

[0177] Metabolic glycoengineering (MGE) manipulates cellular metabolism to regulate glycosylation MGE can be used to increase the levels of natural glycans as well as to substitute unnatural monosaccharides into complex polysaccharides (Agatemor et al., Natl. Acad. Sci., 2013). Rev Chem 3:605-620(2019)). For example, the use of MGE can be used to supply metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) to the sialic acid biosynthetic pathway, resulting in unnatural sialoside display (Du et al., 2009, Glycobiology 19(12):1382-401).

[0132]

[0178] Certain non-mammalian (e.g., plant) proteins are included in the serum proteins of the present disclosure. For example, the cell culture system may include a component that includes serine protease inhibitor activity, but the soybean trypsin inhibitor is not the at least one plant protein homolog. Similarly, the soybean-based antioxidant is not a serum protein homolog. Thus, in some embodiments, the at least one plant protein homolog is not a trypsin inhibitor. In some embodiments, the at least one plant protein homolog is not a soybean-based antioxidant.

[0133]

[0179] Plant protein homologs can be obtained from a number of sources.

[0180] In some embodiments, the plant protein homolog is at least one of a homologue of a serum protein. It may be any plant extract or fraction of a plant extract that contains one plant protein homologue. By way of non-limiting example, the at least one plant protein homologue may be from or in the water-soluble fraction of a plant protein extract. The water-soluble fraction of the plant protein isolate may include plant albumin and plant globulin. In other embodiments, the water-soluble fraction of the plant protein isolate may include plant albumin. Methods for obtaining plant extracts and fractions are known in the art.

[0134]

[0181] The plant protein extract may contain one or more plant protein homologues. For example, in some embodiments, the plant protein isolate may include plant albumins and plant globulins. In other embodiments, the plant protein isolate may include plant albumins.

[0135]

[0182] In some embodiments, the plant protein homolog is isolated from a plant extract. Isolation of plant proteins is well known to those of skill in the art and includes, by way of non-limiting examples, liquid fractionation, centrifugation, column exchange, all of which are routine experimentation.

[0136]

[0183] Plant protein homologs can be purified from plant extracts. In embodiments, at least one plant protein homologue may be in a pure form.

[0184] In another embodiment, the plant protein homolog is derived from a plant extract or plant isolate. , and are not purified to their pure form. Rather, a plant extract or isolate containing at least one plant protein homologue is used as a source of at least one plant protein homologue. Thus, in some embodiments, the at least one plant protein homologue may be in the form of a plant extract fraction. The plant fraction may be processed or further separated into an isolate or further fraction. In some embodiments, the plant fraction or isolate may be concentrated.

[0137]

[0185] In some embodiments, at least one plant protein homologue of a serum protein is The homologues are recombinantly produced. Methods for recombinant production of plant proteins are known to those of skill in the art. Once produced, in some embodiments, the recombinant plant protein homologues can be produced by recombinant methods known to those of skill in the art. The peptides may be isolated and / or purified by methods known in the art.

[0138]

[0186] Yet another aspect of the present disclosure is a method for producing a cell culture medium supplement comprising: A kit is provided that includes a serum-free medium and instructions for mixing the supplements. In some embodiments, the serum-free medium lacks any animal protein. In some embodiments, the serum-free medium lacks any animal components. The kit may further include additional components for cell culture.

[0139]

[0187] Further aspects of the present disclosure include the use of alternative animal proteins in cell culture media supplements. Alternatively, the use of a plant protein homologue of an animal protein is provided. In some embodiments, the animal protein is a serum protein. In some embodiments, the supplement lacks any animal protein. In some embodiments, the supplement lacks any animal component. The plant protein homologue can be one or more of those disclosed herein.

[0140]

[0188] In one aspect of the present disclosure, the animal cell or tissue growth promoting activity of serum homologues is measured. In some embodiments, the source of cells or tissues is any edible species desirable for consumption, including, but not limited to, livestock, poultry, fish, shellfish, crustaceans, and mollusks.

[0141]

[0189] In some embodiments, the source of the cells or tissue is livestock, e.g., cattle, sheep, In some embodiments, the source of the cells or tissue is a crustacean, such as a pig, goat, ram, horse, donkey, rabbit, and mule. In some embodiments, the source of the cells or tissue is an animal traditionally considered to be a "game animal", such as caribou, bear, wild boar, deer, elk, and moose. In some embodiments, the source of the cells or tissue is poultry, such as chicken, duck, goose, guinea fowl, quail, and turkey. In some embodiments, the source of the cells or tissue is fish, such as bass, carp, catfish, Patagonian toothfish, cod, flounder, halibut, mahi-mahi, monkfish, pike, perch, orange roughy, salmon, herring, snapper, swordfish, tilapia, trout, and tuna. In some embodiments, the source of the cells or tissue is crustacean, such as crab, crayfish, lobster, prawn, and shrimp. In some embodiments, the source of cells or tissues is a mollusc, such as bivalve mollusks, mussels, octopus, oysters, scallops, and squid.

[0142]

[0190] In some embodiments, the assays involve the use of serum homologs as substitutes for certain media components. In some embodiments, the assay is designed to determine whether a serum homologue increases cell proliferation and / or density when used as a supplement to a given medium, and the serum homologue is tested by adding it to a growth medium in which one or more given components of the medium have been reduced, removed, or not added. In some embodiments, the assay is designed to determine whether a serum homologue increases cell proliferation and / or density when used as a supplement to a given medium.

[0143]

[0191] In some embodiments, the assay system comprises an animal cell or tissue and an animal cellular In some embodiments, the medium comprises a medium suitable for the growth and / or development of an animal cell or tissue. In some embodiments, the medium comprises components in sufficient amounts for the growth of an animal cell or tissue. In some embodiments, the medium comprises most but not all components in sufficient amounts for the growth of an animal cell or tissue. In some embodiments, the medium is serum-free. In some embodiments, the medium comprises certain serum components but lacks other serum components. In some embodiments, one or more serum components are reduced, subtracted or removed, for example, by immunological (e.g., antibody) means.

[0144]

[0192] In some embodiments, the medium is adapted for use as a serum-free complete medium. In addition, the medium or liquid base mixture may contain one or more components of a basal medium and supplements, such as salts, amino acids, vitamins, buffers, nucleotides, antibiotics, etc., as described herein. The supplement may contain nutrients, trace elements, antioxidants and glucose or an equivalent energy source.

[0145]

[0193] Exemplary inorganic salts include, but are not limited to, potassium phosphate, calcium chloride, Exemplary organic salts include, but are not limited to, sodium bicarbonate or HEPES.

[0146]

[0194] Exemplary sugars include, but are not limited to, dextrose, glucose, lactose, and glycerol. Examples of sugars that can be used include saccharides such as saccharides containing saccharide units such as saccharide units of saccharide groups, ...

[0195] Exemplary antioxidants include, but are not limited to, tocopherol, tocotrienol, Tocopherol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocopherol quinone, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, beta-carotene, selenium, ubiquinone, lutein, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, citric acid, L-carnitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cystathionine, and glycine-glycine-histidine (a tripeptide).

[0147]

[0196] Exemplary trace elements include, but are not limited to, copper, iron, zinc, manganese, potassium, and the like. Examples of trace elements include iodine, molybdnate, molybdenum, vanadium, nickel, tin, aluminum, silver, barium, bromine, cadmium, cobalt, chromium, calcium, divalent cations, fluorine, germanium, iodine, rubidium, zirconium, or selenium. Further trace elements are disclosed in WO 2006 / 004728.

[0148]

[0197] In some embodiments, the medium or liquid base mixture contains an iron source or an iron transporter. Exemplary iron sources include, but are not limited to, ferric and ferrous salts, such as ferrous sulfate, ferrous citrate, ferric citrate, ferric nitrate, ferric sulfate, ferric ammonium compounds, such as ferric ammonium citrate, ferric ammonium oxalate, ferric ammonium fumarate, ferric ammonium malate, and ferric ammonium succinate. Exemplary iron transporters include, but are not limited to, transferrin and lactoferrin.

[0149]

[0198] In some embodiments, the medium or liquid base mixture contains a copper source or a copper transporter. Exemplary copper sources include, but are not limited to, copper chloride and copper sulfate.

[0150]

[0199] In some embodiments, the iron source or copper source is about 0.05 to 250 ng / ml, 0. It is further contemplated that the iron source or copper source is added to the serum replacement medium at a final concentration in the range of about 0.05, 0.1, 0.25, 0.35, 0.45, 0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 ng / ml of serum replacement.

[0151]

[0200] In some embodiments, serum replacement or media supplements are added to the basal medium. Standard basal media are known in the art of cell culture and are commercially available. Examples of basal media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), DMEM F12 (1:1), Iscove's Modified Dulbecco's Medium, Ham's Nutrient Mixture F-10 or F-12, Roswell Park Memorial Institute Medium (RPMI), MCDB131, Click's Medium, McCoy's 5A Medium, Medium 199, William's Medium E, and insect media such as Grace's Medium and TNM-FH.

[0152]

[0201] The serum replacements and media supplements described herein are commercially available serum-free culture media. Also contemplated for use in culture media. Exemplary serum-free media include, but are not limited to, AIM-V (Life Technologies, Carlsbad, Calif.), PER-C6 (Life Technologies, Carlsbad, Calif.), Knock-Out™ (Life Technologies), StemPro® (Life Technologies), CellGro® (Corning Life Sciences-Mediumtech Inc., Manassas, VA.).

[0153]

[0202] Any of these media may optionally contain salts (sodium chloride, calcium, magnesium The medium is supplied with nutrients such as calcium, potassium, and phosphate, amino acids, vitamins, buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), antioxidants, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. will be apparent to one of skill in the art.

[0154]

[0203] It is contemplated that the media composition may be packaged in unit form. In one embodiment, The media (serum replacement, media supplement, complete media or cryopreservation media) are packaged in volumes of 10 ml, 50 ml, 100 ml, 500 ml or 1 L.

[0155]

[0204] In one aspect of the disclosure, the cell culture medium supplement and / or medium disclosed herein The present invention provides a method for culturing cells, comprising:

[0205] Media, e.g., serum replacements, media supplements, complete media, as described herein are useful for culturing cells in vitro, preferably cells that typically require serum replacement or chemically defined media for adequate growth in vitro. Such cells include eukaryotic cells such as mammalian cells and insect cells. Mammalian cells that may benefit from the use of serum replacement, complete media or media supplements include, but are not limited to, hamster, monkey, chimpanzee, dog, cat, cow / bull, pig, mouse, rat, rabbit, sheep and human cells. Insect cells include cells from Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori.

[0156]

[0206] Cells cultured with serum replacement, complete medium or media supplements are immortal. It is contemplated that the cells that are immortalized (cell lines) or non-immortalized (primary or secondary) cells may be any of a wide variety of cell types found in vivo. Exemplary cell types include, but are not limited to, fibroblasts, keratinocytes, epithelial cells, ovarian cells, endothelial cells, glial cells, neural cells, blood forming elements (e.g., lymphocytes, bone marrow cells), chondrocytes and other bone-derived cells, hepatocytes, pancreatic, and precursors of these somatic cell types.

[0157]

[0207] In some embodiments, cells contemplated for use with the medium are derived from a mammalian subject. Cells isolated from a mammalian subject include, but are not limited to, pluripotent stem cells, embryonic stem cells, bone marrow stromal cells, hematopoietic progenitor cells, immune stem cells, bone marrow stem cells, lymphocytes, T cells, B cells, macrophages, endothelial cells, glial cells, neural cells, chondrocytes and other bone-derived cells, hepatic cells, pancreatic cells, precursors of somatic cell types, and any carcinoma or tumor-derived cells.

[0158]

[0208] In some embodiments, the cell is a cell line. Exemplary cell lines include, but are not limited to, Examples of cells that may be used to express the IgG1 gene include, but are not limited to, Chinese hamster ovary cells, including CHOK1, DXB-11, DG-44, and CHO / -DHFR; monkey kidney CV1, COS-7; human embryonic kidney (HEK) 293; baby hamster kidney cells (BHK); mouse Sertoli cells (TM4); African green monkey kidney cells (VERO); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatoma cells (HepG2; SK-Hep); mouse mammary tumor (MMT); TRI cells; MRC 5 cells; FS4 cells; T cell lines (Jurkat); B cell lines, mouse 3T3, RIN, A549, PC12, K562, PER.C6.RTM., SP2 / 0, NS-0, U20S, HT1080, L929, hybridomas, tumor cells, and immortalized primary cells.

[0159]

[0209] Exemplary insect cell lines include, but are not limited to, Sf9, Sf21, HIG These include H FIVE.TM., EXPRESSF+.RTM., S2, Tn5, TN-368, BmN, Schneider2, D2, C6 / 36 and KC cells.

[0160]

[0210] Further cell types and cell lines are described in International Publication No. 2005 / 013363, which is incorporated herein by reference. These cells include, but are not limited to, CD34+ hematopoietic stem cells and cells of myeloid lineage, 293 embryonic kidney cells, A-549, Jurkat, Namalwa, Hela, 293BHK cells, HeLa cervical epithelial cells, PER-C6 retinal cells (PER.C6), MDBK (NBL-I) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLa S3 cells, Hep-G2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-I cells, YI cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, TH-I, B1 cells, or derivatives thereof, any tissue or organ (including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph nodes, adenoids, tonsils, bone marrow, and blood), spleen, fibroblasts and fibroblast-like cell lines), TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit551 cells, De troit510 cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, MR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiC11 cells, CV-I cells, COS-I cells, CO S-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-I1 cells, NOR-IO cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, mouse L cells, Strain 2071 (mouse L) cells, LM line (mouse L) cells, L-MTK (mouse L) cells. These include cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, NSO, NS1, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, Jensen cells, COS cells and Sp2 / 0 cells, mimic cells and / or derivatives thereof.

[0161]

[0211] The cell culture conditions contemplated herein include any culture conditions suitable for growing cells. The cells may be applied to a substrate. Substrates having suitable surfaces include tissue culture wells, culture flasks, roller bottles, gas permeable vessels, flat or parallel plate bioreactors or cell factories. Culture conditions in which cells are attached to microcarriers or particles that are maintained in suspension in a stirred tank are also contemplated.

[0162]

[0212] Cell culture methods are usually described in Culture of Animal Cells: A Manual of Basic Technique, 6th Edition, 2010 (RI Freshney ed., Wiley & Sons); General Techniques of Cell Culture (MA Harrison & IF Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Other reference texts are Creating a High Performance Culture (Aroselli, Hu. Res. Dev. Pr. 1996) and Limits to Growth (DH Meadows et al., Universe Press). Publ. 1974. Tissue culture supplements and reagents are well known to those skilled in the art and are commercially available.

[0163]

[0213] Serum replacement, complete medium or medium supplements may be used with the particular cell line It will be understood that the cells are placed in culture at a density appropriate for the cell type being isolated. In some embodiments, the cells are at a density of 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 Or 5 x 10 6 The cells are cultured at 100 cells / ml.

[0164]

[0214] In some embodiments, the cultured cells are fibroblasts. are bovine fibroblasts. In one embodiment, the cells are chicken fibroblasts.

[0215] Chicken embryo fibroblasts are widely used in the production of viruses and vaccines. Along with chicken embryonic liver cells, they are produced from specific pathogen-free (SPF) embryos and are sold by Charles River Laboratories (Wilmington, MA) and other companies. Chicken liver cells, like their mammalian counterparts, show limited proliferation in culture, but chicken fibroblasts can undergo more than 30 population doublings, producing approximately 2.6 tonnes of cells before spontaneously immortalizing without tumorigenesis. Naturally transformed chicken fibroblasts, such as UMNSAH / DF-1 (CRL-12203), can be purchased directly from ATCC (Manassas, VA). Although the proliferation potential of fibroblasts is excellent, the cells form primarily inedible connective tissue.

[0165]

[0216] Chick embryo endothelium can be easily isolated, but their proliferation potential is unknown and It may be organ-specific. Mouse microvascular cells can undergo 30 population doublings, whereas human cells rarely exceed 12 population doublings. Chick embryonic muscle cells (myocytes) can be similarly isolated, but have very limited proliferative capacity. Mouse and human cells rarely exceed 12 population doublings. Myogenesis, the formation of new muscle tissue, is an uncommon process during the neonatal period of life in most species. Small molecules could conceptually be used to modulate this behavior.

[0166]

[0217] Over the last decade, many groups have developed methods to generate chicken embryonic stem cells (cESCs). The cells are isolated from fertilized chicken eggs and are essentially immortal. Chicken induced pluripotent stem cells (ciPSCs) have been produced from quail embryo fibroblasts with the reprogramming factors OCT4, NANOG, SOX2, LIN28, KLF4, and C-MYC, and more recently from chicken fibroblasts using OCT4, KLF4, and C-MYC. The cells are essentially immortal, but have been genetically engineered.

[0167]

[0218] Recently, mouse pluripotent stem cells have been shown to generate muscle, hepatic, and endothelial cells as well as complex germ layers. They have been derived from fibroblasts using small molecules that allow for the differentiation of multiple cell types, including myeloid leukemia (MLH) and myocardial infarction (MCI). Chemical induction of ciPSCs offers an alternative approach to converting fibroblasts into other cell types.

[0168]

[0219] More recent studies have identified nine proteins that induce human fibroblasts to transform into cardiomyocytes. A combination of compounds was identified, whereas others used a combination of seven compounds to transform mouse cells. Given that many signaling pathways are conserved, relatively similar combinations can be used to transform chicken fibroblasts into muscle cells.

[0169]

[0220] As mentioned above, cell culture media contain a variety of nutrients, including attachment factors, fatty acids, growth factors, hormones, and Human serum often contains fetal bovine serum (FBS), which provides the necessary nutrients and albumin. FBS can be replaced with serum substitutes (e.g., KO serum), which are typically composed of transferrin, insulin, and lipid-rich bovine serum albumin plus amino acids, vitamins, and trace elements. Both transferrin and insulin are produced in bacteria using recombinant technology, while albumin is usually of animal origin. However, plant and bacterial derived recombinant human albumin (e.g., Cellastim™) is available from several companies, including Sigma-Aldrich (St. Louis, MO).

[0170]

[0221] Chicken fibroblast culture media is traditionally made up of 10% FBS, tryptose The medium consists of MI 99 medium supplemented with 0.5 mg / mL albumin, 0.6 μM linolenic acid, 0.6 μg / mL lecithin, 5 ng / niL bEGF, 5 ng / niL EGF, 30 pg / mL TGFpi, 7.5 mM glutamine, 1 μg / mL hydrocortisone, 50 μg / mL ascorbic acid, and 5 μg / mL insulin. This medium, PSC-201-040, is available from ATCC (Manassas, VA) and has been reported to support four times faster growth of human fibroblasts. Chicken hepatocytes are similarly supported by serum-free culture media designed for human and mouse hepatocytes. The culture medium consists of Williams E basal medium supplemented with albumin, insulin, transferrin, and hydrocortisone.

[0171]

[0222] Chicken and bovine anchorage-independent fibroblasts were differentiated by standard differentiation protocols. FMT-SCF-2 (chicken non-adherent fibroblasts) and FMT-SBF-1 (bovine non-adherent fibroblasts) were grown in adipogenic medium containing 200 μM oleic acid together with PPAR gamma agonists. Both synthetic (rosiglitazone) and natural (pristanic acid) inhibitors were tested.

[0172]

[0223] The perfused culture medium may also contain an oxygen carrier. Hemoglobin-based oxygen carriers are Alternatives include hemoglobin derivatives, either recombinant or chemically modified, encapsulated hemoglobin, or modified (e.g., cross-linked) red blood cells. Alternatives include perfluorocarbon-based alternatives, such as those developed in Nahmias et al. (The FASEB Journal, 20(14):2531-2533).

[0173]

[0224] Normally, primary fibroblasts can undergo limited cell differentiation and therefore require approximately 30 population doublings ( It should be noted that immortalized fibroblast cell lines undergo cellular senescence after, for example, 10 passages. Methods for generating immortalized fibroblast cell lines include genetic engineering by introduction of the telomerase gene, or the SV40, or HPVE6 / E7 genes using known methods.

[0174]

[0225] Other avian fibroblasts, such as duck, goose, and quail fibroblasts, are also suitable. It is possible that...

[0226] Another aspect of the present disclosure is to culture cells in any of the cell culture media disclosed herein. The present invention provides a method for producing cultured meat by culturing cells in a mammalian animal, and a method for producing meat from cultured cells.

[0175]

[0227] In some embodiments, the cells are from a food animal. The object is a livestock animal, such as cattle, sheep, pigs, goats, rams, horses, donkeys, rabbits, and mules. In some embodiments, the animal is an animal traditionally considered a "game animal", such as caribou, bear, wild boar, deer, elk, and moose. In some embodiments, the animal is a poultry, such as chicken, duck, goose, guinea fowl, quail, and turkey. In some embodiments, the animal is a fish, such as bass, carp, catfish, Patagonian toothfish, cod, flounder, halibut, mahi-mahi, monkfish, pike, perch, orange roughy, salmon, herring, snapper, swordfish, tilapia, trout, and tuna. In some embodiments, the animal is a crustacean, such as crab, crayfish, lobster, prawn, and shrimp. In some embodiments, the animal is a mollusc, such as a bivalve, a mussel, an octopus, an oyster, a scallop, and a squid.

[0176]

[0228] In some embodiments, the cell is a fibroblast. In one embodiment, the cell is a fibroblast. Examples of fibroblasts include, but are not limited to, bovine fibroblasts and chicken fibroblasts. In one embodiment, the fibroblasts are bovine fibroblasts. In one embodiment, the fibroblasts are chicken fibroblasts.

[0177]

[0229] Yet another aspect of the present disclosure provides cultured meat produced by the above method. .

[0230] Yet another aspect of the present disclosure is a method for producing a food product that is devoid of any animal protein and / or animal components. A cell culture medium and a method for producing the cell culture medium are provided.

[0178]

[0231] The cell culture medium comprises a serum-free medium and at least one plant protein homologue. The cell culture medium may comprise any of the cell culture medium supplements disclosed herein, including any of the cell culture medium supplements disclosed herein. The cell culture medium may lack any animal components and / or may lack any animal proteins. The at least one plant protein homolog may be a homolog of an animal protein. The at least one plant protein homolog may be a homolog of a serum protein. Examples of such plant protein homologs of serum proteins are disclosed above and herein.

[0179]

[0232] In some embodiments, the serum-free medium is a basal physiological buffer and is free of animal contaminants. , human contaminants, or any antibiotic(s). In some embodiments, the serum-free medium is a basal physiological buffer and lacks any animal proteins. In some embodiments, the serum-free medium is a basal physiological buffer and lacks any animal components.

[0180]

[0233] Exemplary serum-free media include, but are not limited to, AIM-V (Life Technologies). echnologies, Carlsbad, Calif.), PER-C6 (Life Technologies, Carlsbad, Calif.), Knock-Out™ (Life Technologies), StemPro® (Life Technologies), and CellGro® (Corning Life Sciences-Mediumtech Inc., Manassas, VA.).

[0181]

[0234] Any of these media may optionally contain salts (sodium chloride, calcium, magnesium The medium is supplemented with phosphate, phosphates, amino acids, vitamins, buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), antioxidants, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. will be apparent to one of skill in the art.

[0182]

[0235] In some embodiments, the cell culture medium can be used as a serum-free complete medium. To allow for the production of cell culture media, the cell culture medium may contain one or more components and supplements of the basal media described herein, such as salts, amino acids, vitamins, buffers, nucleotides, antibiotics, trace elements, antioxidants, and glucose or an equivalent energy source.

[0183]

[0236] Exemplary inorganic salts include, but are not limited to, potassium phosphate, calcium chloride, Exemplary organic salts include, but are not limited to, sodium bicarbonate or HEPES.

[0184]

[0237] Exemplary sugars include, but are not limited to, dextrose, glucose, lactose, and glycerol. Examples of sugars that can be used include saccharides such as saccharides containing saccharide units such as saccharide units of saccharide groups, ...

[0238] Exemplary antioxidants include, but are not limited to, tocopherol, tocotrienol, Tocopherol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocopherol quinone, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, beta-carotene, selenium, ubiquinone, lutein, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, citric acid, L-carnitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cystathionine, and glycine-glycine-histidine (a tripeptide).

[0185]

[0239] Exemplary trace elements include, but are not limited to, copper, iron, zinc, manganese, potassium, and the like. Examples of trace elements include iodine, molybdnate, molybdenum, vanadium, nickel, tin, aluminum, silver, barium, bromine, cadmium, cobalt, chromium, calcium, divalent cations, fluorine, germanium, iodine, rubidium, zirconium, or selenium. Further trace elements are disclosed in WO 2006 / 004728.

[0186]

[0240] In some embodiments, the cell culture medium comprises an iron source or an iron transporter. Exemplary iron sources include, but are not limited to, ferric and ferrous salts such as ferrous sulfate, ferrous citrate, ferric citrate, ferric nitrate, ferric sulfate, ferric ammonium compounds such as ferric ammonium citrate, ferric ammonium oxalate, ferric ammonium fumarate, ferric ammonium malate, and ferric ammonium succinate. Exemplary iron transporters include, but are not limited to, transferrin, lactoferrin.

[0187]

[0241] In some embodiments, the cell culture medium contains a copper source or a copper transporter (e.g., Exemplary copper sources include, but are not limited to, copper chloride and copper sulfate.

[0188]

[0242] In some embodiments, the iron source or copper source is about 0.05 to 250 ng / ml, 0. The antibody is added to the cell culture medium at a final concentration in the range of about 0.05-100 ng / ml, about 0.05-50 ng / ml, about 0.05-10 ng / ml, about 0.1-5 ng / ml, about 0.5-2.5 ng / ml, or about 1-5 ng / ml.

[0189]

[0243] It is contemplated that the cell culture medium is packaged in a unit form. In the present invention, cell culture media is packaged in volumes of 10 ml, 50 ml, 100 ml, 500 ml or 1 L.

[0190]

[0244] The cell culture medium may further comprise other components, said components being any animal component. and / or may lack animal protein.

[0245] Methods for producing the cell culture media described herein are also disclosed. The method of producing the culture medium may include combining a serum-free basal medium and a cell culture medium supplement, the cell culture medium lacking any animal protein and / or animal components. The cell culture medium supplement includes at least one plant protein homolog of an animal protein. In some embodiments, the animal protein is a serum protein. The plant protein homolog may be one or more of those disclosed herein.

[0191]

[0246] Exemplary serum-free media are provided herein.

[0247] In some embodiments, the method further comprises the step of adding one or more additional ingredients. The ingredients may further comprise a top up, said ingredients being devoid of any animal components. Exemplary additional ingredients are provided herein.

[0192]

[0248] The present disclosure includes the cell culture media described herein and instructions for use. Kits are further provided. In some embodiments, the cell culture medium is packaged in a container with a label attached thereto or contained in a package that describes the use of the composition for in vitro, in vivo, or ex vivo use. Exemplary containers include, but are not limited to, containers, vials, tubes, ampoules, bottles, flasks, and the like. It is further contemplated that the container is suitable for packaging culture medium in liquid or frozen form, such as serum replacement, medium supplement, or cryopreservation medium. It is contemplated that the container is made from materials known in the art, including, but not limited to, glass, polypropylene, polystyrene, and other plastics. In various aspects, the composition is packaged in a unit dosage form. The kit optionally includes a device suitable for combining the serum replacement, medium supplement, or cryopreservation medium with the basal medium, or combining the medium with additional growth factors. In various aspects, the kit contains a label and / or instructions that describe the use of the medium for cell culture or cryopreservation.

[0193]

[0249] All applications and all documents cited herein or pending thereon ("Applications") All documents cited or referenced in the documents cited in the application, and all documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products described in this specification or any documents incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present invention. More particularly, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0194]

[0250] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0195] Example 1: Isolation and purification of plant albumins

[0251] Isolation of plant albumins is well known to those skilled in the art and can involve techniques such as liquid fractionation, centrifugation, column chromatography, and the like. All of this is routine experimentation, including exchanges. In this study, two varieties of potatoes (white and red) were purchased. Each variety was either juiced (juicer) or blended (blender). Potato albumin was extracted and the liquid fraction of raw white and red potatoes was extracted using either a fruit juicer or a kitchen blender. Afterwards, the potatoes were chopped and added to the blender with excess water. The blended material was sieved / filtered using two layers of gauze to obtain the liquid fraction. The samples were spun down at 11000 rpm for 10 minutes at 4°C to remove insoluble material. The supernatant (albumin fraction) was collected and the pellet was discarded.

[0196]

[0252] The supernatant samples were prepared according to the method described by Jirgensons (1946, Journal of Po The pH was adjusted to 3 according to the method of Example 1 (Lymer Science, 1(6):484-494), and then spun down again to remove insoluble protein impurities. The supernatant was dialyzed overnight against 20 mM TRIS-HCl, pH 8. To remove excess salt in the samples, each was dialyzed against buffer A (see below). 1 lit for 1 hour, then 1 lit for 2 hours. ON (cold room, with strearing).

[0197]

[0253] The four samples were loaded onto an anion exchange column according to pI ~5 and albumin was Fractions were separated. Sample loading buffer was 20 mM TRIS HCl, pH 8, and release buffer was 20 mM TRIS HCl, 1 M NaCl, pH 8. 8 μl samples from the extracted fractions were loaded onto a 4-20% SDS-PAGE for protein purity display and analysis (Figure 3F), with 1% BSA loaded as a control. Appropriate fractions were combined and several μg of each were subjected to MS analysis to confirm the BSA-like protein content (Figures 3A-3E).

[0198]

[0254] Five grams of pea protein isolate was added to the sample prepared according to the method described by Nadal et al., JA According to the method of Food Chem. 2011, 59, 2752-2758, the samples were suspended in 50 ml of 10 mM CaCl2, 10 mM MgCl2, pH 8. The samples were vortexed for 30 min at room temperature, then spun down at 11000 rpm for 15 min at 4°C, and the supernatants were collected and subjected to SDS-PAGE (Figure 4).

[0199]

[0255] Five plant flours (durum, chickpea, lentil, corn, rice) Chickpea and two commercial plant protein isolates (hemp, pea) were liquid fractionated to separate lipids from DNA and RNA, as well as from protein. The aqueous fractions of the protein extracts were collected and subjected to SDS-PAGE. Chickpea, corn, hemp and pea samples contain protein bands corresponding to the previously reported size of albumin protein (Figure 5). The boxed bands were isolated from the gel and sent for MS analysis for further identification.

[0200]

[0256] MS analysis of four potato extracts from two potato types (red or white) The results are shown in Figure 6. Each was either liquefied in a fruit juicer (J) or blended with water (B) and sieved to remove the fiber fraction. BSA was analyzed as a positive control. All potato samples were shown to contain significant amounts of BSA-like proteins. Identified proteins in potato (Solanum tubrosum) included albumin (patatin) (UniProtKB: M1AGX5, Q2MYP6, Q2VBI2, Q2VBJ3, A0A097H149), a patatin-like phospholipase domain-containing protein. Proteinase inhibitors (Kunitz-type proteinase inhibitor group A1) (UniProtKB: H9B8I9); 20 kDa Kunitz-type proteinase inhibitor (UniProtKB: Q9S8K2).

[0201] Example 2: Adhesion of cultured cells in serum-free medium

[0257] Soluble plant ECM-like proteins are in the absence of serum and animal-derived ECM proteins. It was hypothesized that the presence of 100% glycerol in the extracts would be able to support the adhesion of cultured cells. Protein extraction from chickpea, lentil, durum and brown rice flours was performed by suspending them in PBS with shaking for 24 hours at room temperature, spinning down at 13000×g and filtering using a 0.22 μm syringe filter. Primary chicken fibroblasts cultured in DMEM / F12 supplemented with 15% FBS were trypsinized, washed and re-seeded in serum-free medium supplemented with 1:50 or 1:100 dilutions of the protein constructs described above. The extent of adhesion was assessed after 8 hours using Sulforhodamin B staining (Vichai and kirtikara, Nat Protoc. 2006;1(3):1112-6). The results demonstrate cell adhesion in soybean, chickpea, lentil, rice and wheat extracts (Figure 7).

[0202] Example 3: Preparation of complete protein bulk

[0258] To prepare complete protein bulk as an alternative to bovine serum albumin (BSA) To determine the protein content, protein powders from different plant sources were first mixed with water or saline (PBS) in a stirrer. The % protein recovery was measured for the different plant protein sources and ranged from 10% to 15%. The mixture was then centrifuged using a Sorval at high speed to remove the insoluble fraction, and then the soluble fraction was filtered and concentrated using either a Centricon, Amplicon, or Hollow Fiber with a cut-off of 10 kilodaltons (Figure 8). The mixture was then stored at +4°C and used for a period of 1-2 months.

[0203] Example 4: Albusorb purification of soy proteins

[0259] To purify soybean protein (soluble fraction), 50 mg of AlbuSor The AlbuSorb™ powder was placed in a spin tube / microtube. 400 μl of Binding Buffer BB1 was added to the tube to prepare the AlbuSorb™ powder. After mixing the contents either manually or by vortexing for 3 minutes, the tube was then centrifuged at 3000 rpm for 2 minutes. The supernatant was discarded. Another 400 μl of BB1 buffer was again added to the tube, then mixed and centrifuged. The supernatant was again discarded.

[0204]

[0260] For albumin binding requirements, 250 μl of BB1 buffer was added, followed by 25 μl of serum was added at 1000 rpm. The tubes were then placed on a rotary shaker for 10 minutes. Afterwards, the tubes were centrifuged at 10000 rpm for 4 minutes. The resulting supernatant contains serum proteins free of albumin. Optionally, the pellet (mostly albumin) can be eluted with 200 μl of stripping buffer (0.2 M Tris + 0.5 M NaCl, pH 10) by mixing on a rotary shaker for 10 minutes and centrifuging at 10000 rpm for 4 minutes.

[0205]

[0261] The supernatant was collected and subjected to SDS-PAGE. The soybean proteins (water-soluble fraction) after digestion are shown. When observing the proteins on SDS-PAGE (4-15%), it is possible that other proteins migrate in the same region as albumin and may not have been completely digested.

[0206]

[0262] The water-soluble soy protein and the Albusorb purified soy protein further The samples were sent for MS analysis for further identification. The top 10 protein groups in the soybean water-soluble fraction before and after rb purification are shown.

[0207] Example 5: MS analysis of chickpea proteins

[0263] 8M urea, 25mM Tris-HCl, pH 8.0, 10mM dithiothreitol Ten micrograms of chickpea protein dissolved in diethyl ether (DTT) was alkylated with 55 mM iodoacetamide for 30 min at room temperature. Samples were diluted 8-fold with Tris-HCl, pH 8.0. 0.3 μg trypsin (sequencing grade, from Promega Corp., Madison, WI, USA) was then added to the samples and digestion was carried out overnight at 37°C. Tryptic peptides were desalted on a C18 Stage tip (Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc. 2007;2(8):1896-906). A total of 0.8 μg peptides (by OD280nm) were injected into mass spectrometry (MS) for analysis (Figure 11).

[0208]

[0264] MS analysis was performed online using a nanoflow UHPLC system (Ultimate 3 The analysis was carried out using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to a 1000 000 Dionex, Thermo Fisher Scientific). Eluted peptides were separated on a 90 min gradient run on a reversed phase 25 cm-long C18 column (75 um ID, 2 um, 100 Å, Thermo PepMap® RSLC) at a flow rate of 0.2 μl / min. A survey scan (380-2000 m / z, target value 3E6 charge, maximum ion injection time 200 ms) was acquired followed by higher energy collision dissociation (HCD) based fragmentation (normalized collision energy 25). A resolution of 70000 was used for the survey scan to fragment up to 15 kinetically selected most abundant precursor ions (isolation window 1.6 m / z). MS / MS scans were acquired at a resolution of 35000 (target value 2E5 charge, maximum ion injection time 121 ms). Dynamic exclusion was 15 seconds.

[0209]

[0265] MS data were analyzed using MaxQuant computational platform orm,version 1.6.6.0(Cox,J. & Mann,M.MaxQuant enables high peptide identification The peaks were processed using the Uniprot Cicer arietinum database, which contains 57497 entries. The search included carbamidomethylation of cysteine ​​as fixed modification and oxidation of methionine and N-terminal acetylation as variable modifications. Peptides with a minimum length of 7 amino acids were considered and the required FDR was set at 1% at peptide and protein level. Protein identification required at least two unique or razor peptides.

[0210] Example 6: Plant proteins replacing bovine serum albumin

[0266] The effect of different plant water-soluble fraction proteins was investigated using a special serum-free medium depleted of BSA. Suspension culture-adapted chicken fibroblasts were seeded at 300,000 cells / ml in a flask with a total volume of 20 ml. Cell culture flasks were maintained in a shaking incubator at 100 rpm, 39°C, and 5% CO2. On day 3, a 1 ml sample from each flask was counted using an automated cell counter (APOI) (C ellaca) to determine live from dead cells. The viable cell count is shown in Figure 12. It was shown that 0.1 mg / ml was sufficient to replace the animal protein (BSA). However, hemp and wheat proteins did not support the proliferation of chicken fibroblasts at this concentration.

[0211] Example 7: Plant proteins and dose-dependent effects

[0267] Gradient concentrations of both chickpea protein and organic pea protein were grown in suspension medium. It was tested on chicken fibroblasts in culture, replacing animal protein (BSA) in serum-free medium. Chicken fibroblasts adapted to suspension culture were seeded at 300,000 cells / ml in a total flask volume of 20 ml. Cell culture flasks were maintained in a shaking incubator at 100 rpm, 39°C, and 5% CO2. As shown in Figure 13, different ranges of protein concentrations perform almost as well as or better than BSA. Cell counts were performed on day 3 using an automated cell counter (Cellaca) that uses APOI staining to exclude dead cells from our counts.

[0212]

[0268] Increasing concentrations of chickpea or soy protein (data not shown) were associated with increased The results are a cytotoxic effect of the centrilobacterial enzyme (Figure 14). Deletion of the <10 kilodalton fraction from the protein mixture using Centricon significantly removes this toxicity. Suspension culture-adapted chicken fibroblasts were seeded at 300,000 cells / ml in a total flask volume of 20 ml. Cell culture flasks were maintained in a shaking incubator at 100 rpm, 39°C, and 5% CO2. On day 5, a 1 ml sample from each flask was counted using an automated cell counter (Cellaca) using AOPI to determine live from dead cells. The viable cell counts are shown in Figure 14.

[0213]

[0269] The gradient concentration of chickpea is a hollow fiber with a cutoff value of 10 kilodaltons. The cells were washed three times with 500 mM NaCl. Chickpea at 5 mg / ml was not toxic to the chicken cells (Figure 15). Suspension culture-adapted chicken fibroblasts were seeded at 0.3 million cells / ml in a flask with a total volume of 20 ml. Cell culture flasks were maintained in a shaking incubator at 100 rpm, 39°C, and 5% CO2. On days 3-6, 1 ml samples from each flask were counted using an automated cell counter (Cellaca) using AOPI to determine live from dead cells. The viable cell counts are shown in Figure 15.

[0214]

[0270] Although the present invention and its advantages have been described in detail, the spirit and scope of the invention as defined in the appended claims is not to be construed as limiting the present invention. It should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope.

[0215]

[0271] Those skilled in the art will appreciate that the present invention carries out the objects and is not limited to those described and intended by the present invention. It will be readily understood that the present invention is fully adapted to obtain the objects and advantages thereof. The present examples along with the methods described herein are presently representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art and are encompassed within the scope of the invention as defined by the scope of the claims.

Claims

1. A cell culture medium supplement comprising at least one plant protein homologue of a serum protein.

2. 10. The cell culture media supplement of claim 1, wherein the supplement lacks any serum proteins.

3. 3. The cell culture medium supplement of claim 1 or 2, wherein the supplement is essentially devoid of any animal serum derived components.

4. 4. The cell culture medium supplement of any one of claims 1 to 3, wherein the at least one plant protein homologue comprises a water soluble fraction of a plant protein isolate.

5. 5. The cell culture medium supplement of claim 4, wherein the water soluble fraction comprises plant albumins and globulins.

6. 4. The cell culture medium supplement of any one of claims 1 to 3, wherein the at least one plant protein homolog is a homolog of serum albumin, serum catalase, serum superoxide dismutase, serum transferrin, serum fibronectin, serum vitronectin, serum insulin, serum hemoglobin, serum aldolase, serum lipase, serum transaminase, serum aminotransferase, serum fetuin, or combinations thereof.

7. 7. The cell culture media supplement of claim 6, wherein the at least one plant protein homolog is plant albumin, plant catalase, plant superoxide dismutase, plant transferrin, plant fibronectin, plant vitronectin, plant insulin, plant leghemoglobin, plant aldolase, plant lipase, plant transaminase, plant aminotransferase, plant cystatin, or a combination thereof.

8. 8. The cell culture medium supplement of claim 7, wherein the at least one plant protein homolog comprises a plant albumin, a plant catalase, a plant fibronectin, and a plant insulin.

9. 8. The cell culture medium supplement of claim 7, wherein the at least one plant protein homolog is a plant albumin.

10. 10. The cell culture medium supplement of claim 8 or 9, wherein the plant albumin is chickpea albumin, hemp seed albumin, lentil albumin, pea albumin, soybean albumin, wheat albumin or potato albumin.

11. 11. The cell culture medium supplement of claim 10, wherein the plant albumin is pea albumin or potato albumin.

12. 10. The cell culture medium supplement of claim 9, wherein the plant albumin is derived from a water soluble fraction of a plant protein isolate.

13. 13. The cell culture medium supplement of any one of claims 7 to 12, wherein the plant albumin has a molecular weight of about 13 to 110 kilodaltons.

14. 14. The cell culture medium supplement of claim 13, wherein the plant albumin has a molecular weight of about 13-17 kilodaltons.

15. 14. The cell culture medium supplement of claim 13, wherein the plant albumin has a molecular weight of about 20-35 kilodaltons.

16. 14. The cell culture medium supplement of claim 13, wherein the plant albumin has a molecular weight of about 50-110 kilodaltons.

17. 17. The cell culture medium supplement of any one of claims 7-16, wherein the phytoalbumin is present in the cell culture medium supplement at a concentration such that when the cell culture medium supplement is added to a cell culture medium, the phytoalbumin has a final concentration of about 0.01% to about 10% by weight in the cell culture medium.

18. 8. The cell culture media supplement of claim 7, wherein the at least one plant protein homolog is a plant catalase.

19. 20. The cell culture medium supplement of claim 8 or 18, wherein the plant catalase is Arabidopsis catalase, cabbage catalase, cucumber catalase, cotton catalase, potato catalase, pumpkin catalase, spinach catalase, sunflower catalase, tobacco catalase or tomato catalase.

20. 20. The cell culture media supplement of claim 19, wherein the plant catalase is cabbage catalase, cucumber catalase, or potato catalase.

21. 21. The cell culture medium supplement of any one of claims 18-20, wherein the plant catalase has a molecular weight of about 50-70 kilodaltons.

22. 22. The cell culture medium supplement of any one of claims 18-21, wherein the plant catalase is present in the cell culture medium supplement at a concentration such that when the cell culture medium supplement is added to a cell culture medium, the plant catalase has a final concentration of about 1 ng / ml to about 100 ng / ml in the cell culture medium.

23. 8. The cell culture media supplement of claim 7, wherein the at least one plant protein homolog is a plant fibronectin.

24. 24. The cell culture media supplement of claim 8 or 23, wherein the plant fibronectin is bean fibronectin, chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin, tobacco fibronectin or wheat fibronectin.

25. 25. The cell culture media supplement of claim 24, wherein the plant fibronectin is chickpea fibronectin, lentil fibronectin, rice fibronectin, soybean fibronectin or wheat fibronectin.

26. 26. The cell culture medium supplement of any one of claims 23 to 25, wherein the plant fibronectin has a molecular weight of about 40 to 60 kilodaltons.

27. The plant fibronectin is at about 0.1 μg / ml to about 100 μg / ml in the cell culture medium when the cell culture medium supplement is added to the cell culture medium.

27. The cell culture medium supplement of any one of claims 23 to 26, wherein the cell culture medium supplement is present in said cell culture medium supplement at a concentration having a final concentration of 100 mg / ml.

28. 8. The cell culture medium supplement of claim 7, wherein the at least one plant protein homolog is a plant insulin.

29. 30. The cell culture medium supplement of claim 8 or 28, wherein the plant insulin is glucokinin, charantin, or corosolic acid.

30. 30. The cell culture medium supplement of claims 28 or 29, wherein the plant insulin is present in the cell culture medium supplement at a concentration such that when the cell culture medium supplement is added to a cell culture medium, the plant insulin has a final concentration in the cell culture medium of about 0.05 μg / ml to about 10 μg / ml.

31. 31. The cell culture medium supplement of any one of claims 1 to 30, wherein the at least one plant protein homologue is in the form of a plant extract fraction or in pure form.

32. 32. A cell culture medium comprising a serum-free medium and the cell culture medium supplement of any one of claims 1 to 31, wherein said serum-free medium is essentially devoid of any animal serum-derived components.

33. 33. The cell culture medium of claim 32, wherein the serum-free medium is a basal physiological buffer.

34. 32. A kit comprising the cell culture medium supplement of any one of claims 1 to 31 and instructions for mixing said cell culture medium supplement with a serum-free medium lacking any animal serum-derived components.

35. 34. A method of producing cultivated meat, comprising culturing cells in the cell culture medium of claim 32 or 33, and producing meat from the cultured cells.

36. 36. The method of claim 35, wherein the cells are from a food animal.

37. 37. The method of claim 36, wherein the food animal is a livestock animal, a game animal, a poultry animal, a fish, or a crustacean.

38. The method of any one of claims 35 to 37, wherein the cells are fibroblasts.

39. 39. The method of claim 38, wherein the fibroblasts are bovine fibroblasts or chicken fibroblasts.

40. Cultured meat produced by the method of any one of claims 35 to 39.

41. 32. A method for producing a cell culture medium lacking any animal proteins and / or components, the method comprising the step of mixing a serum-free basal medium with a cell culture medium supplement according to any one of claims 1 to 31, wherein said serum-free basal medium and the cell culture medium supplement each essentially lack any animal serum-derived components.

42. 42. A cell culture medium produced by the method of claim 41.

43. Plant proteins to replace animal proteins in cell culture media supplements Use of protein homologues.

44. 44. The use according to claim 43, wherein the animal protein is a serum protein.

45. 45. The use of claim 43 or 44, wherein the supplement is essentially devoid of any animal serum derived components.

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