Method for culturing animal cells

Abstract

Provided is a method for culturing animal cells in which animal cells are cultured in the presence of an iron ionophore such as hinokitiol. Also provided is technology related to the method.

Description

Method for culturing animal cells

[0001] The present invention relates to a method for culturing animal cells and related techniques.

[0002] Culture media containing serum, such as fetal bovine serum, are widely used for culturing animal cells. However, from the viewpoints of safety and economy, the development of serum-free culture media is desired. For example, serum-free culture media in which serum is replaced with alternative components, such as growth factors, have been developed.

[0003] Transferrin is sometimes used in animal cell culture (Non-Patent Document 1). Transferrin is known to bind to iron and contribute to iron transport (Non-Patent Documents 2 and 3).

[0004] Hinokitiol is also known as an iron ionophore, which has the ability to bind to iron ions and increase the membrane permeability of iron ions (Non-Patent Document 4).

[0005] Tatsuma Yao and Yuta Asayama. Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol. 2017 Apr; 16(2): 99-117.Fang, X. et al. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nature Reviews 20, 7-23 (2023).Waldvogel-Abramowski, S. et al. Physiology of Iron Metabolism. Transfus. Med. Hemother. 41, 213-221 (2014).Science;12 May 2017;Vol.356,Issue 6338;p.608-616; doi: 10.1126 / science.aah3862

[0006] An objective of the present invention is to provide a method for culturing animal cells and related techniques.

[0007] The present inventors have discovered that animal cells can be cultured in the absence of serum by using an iron ionophore such as hinokitiol, and have completed the present invention.

[0008] That is, the present invention can be exemplified as follows. [1] A composition for animal cell culture, containing an iron ionophore. [2] The composition (specifically described in [1]) which is a composition for improving the growth and / or promoting the differentiation of animal cells. [3] The composition (specifically described in [1] or [2]) which is a culture medium or a culture medium additive. [4] The composition (specifically described in [3]) wherein the culture medium is a basal medium, a feed medium, or a perfusion medium. [5] The composition (specifically described in any of [1] to [4]) wherein the iron ionophore is at least one component selected from the group consisting of hinokitiol, α-thujaplicin, and γ-thujaplicin. [6] The composition (specifically described in any of [1] to [5]) wherein the iron ionophore is hinokitiol. [7] The composition (specifically described in any of [1] to [6]) further containing iron. [8] The composition (specifically, according to [7]) wherein the iron is an iron salt. [9] The composition (specifically, according to any one of [1] to [8]) wherein the animal is a mammal, bird, fish, or crustacean.

[10] The composition (specifically, according to any one of [1] to [9]) wherein the animal is a meat-producing animal.

[11] The composition (specifically, according to any one of [1] to

[10] ) wherein the animal is a cow, pig, or chicken.

[12] The composition (specifically, according to any one of [1] to [9]) wherein the animal is a human.

[13] The composition (specifically, according to any one of [1] to

[12] ) wherein the cells are muscle stem cells, preadipocytes, kidney-derived cells, or T cells.

[14] The composition (specifically, according to any one of [1] to

[13] ) wherein the composition is substantially free of serum.

[15] The composition (specifically, the composition described in any one of [1] to

[14] ) which is substantially free of animal-derived albumin.

[16] The composition (specifically, the composition described in any one of [1] to

[15] ) which is substantially free of transferrin.

[17] The composition (specifically, the composition described in any one of [1] to

[16] ) which has the function of improving the growth of animal cells and / or the function of promoting the differentiation of animal cells.

[18] A method for producing a product, the method comprising a step of culturing animal cells in the presence of an iron ionophore.

[19] The method (specifically described in

[18] ) wherein the product is cultured meat.

[20] A method for culturing animal cells, the method comprising a step of culturing animal cells in the presence of an iron ionophore.

[21] A method for improving the growth of animal cells, the method comprising a step of culturing animal cells in the presence of an iron ionophore.

[22] A method for promoting differentiation of animal cells, the method comprising a step of culturing animal cells in the presence of an iron ionophore.

[23] The method (specifically described in any of

[18] to

[22] ) wherein the iron ionophore is at least one component selected from the group consisting of hinokitiol, α-thujaplicin, and γ-thujaplicin.

[24] The method (specifically described in any of

[18] to

[23] ) wherein the iron ionophore is hinokitiol.

[25] The method (specifically, described in any one of

[18] to

[24] ) wherein the culture is carried out in the presence of iron.

[26] The method (specifically, described in

[25] ) wherein the iron is an iron salt.

[27] The method (specifically, described in any one of

[18] to

[26] ) wherein the concentration of the iron ionophore in the medium during the culture is 1 nM to 100 μM.

[28] The method (specifically, described in any one of

[18] to

[27] ) wherein the concentration of the iron ionophore in the medium during the culture is 10 nM to 10 μM.

[29] The method (specifically, described in

[23] or

[24] ) wherein the concentration of hinokitiol in the medium during the culture is 1 nM to 2.5 μM.

[30] The method (specifically, described in

[23] or

[24] ) wherein the concentration of hinokitiol in the medium during the culture is 10 nM to 1 μM.

[31] The method (specifically, the method described in

[25] or

[26] ) wherein the iron concentration in the medium during the culture is 200 nM to 500 μM.

[32] The method (specifically, the method described in any of

[18] to

[31] ) wherein the animal is a mammal, a bird, a fish, or a crustacean.

[33] The method (specifically, according to any one of

[18] to

[32] ) wherein the animal is a meat animal.

[34] The method (specifically, according to any one of

[18] to

[33] ) wherein the animal is a cow, pig, or chicken.

[35] The method (specifically, according to any one of

[18] or

[20] to

[32] ) wherein the animal is a human.

[36] The method (specifically, according to any one of

[18] to

[35] ) wherein the cells are muscle stem cells or preadipocytes.

[37] The method (specifically, according to any one of

[18] or

[20] to

[35] ) wherein the cells are kidney-derived cells or T cells.

[38] The method (specifically, according to any one of

[18] to

[37] ) wherein the culturing is carried out in a medium substantially free of serum.

[39] The method (specifically, according to any one of

[18] to

[38] ) wherein the culture is carried out in a medium substantially free of animal-derived albumin.

[40] The method (specifically, according to any one of

[18] to

[39] ) wherein the culture is carried out in a medium substantially free of transferrin.

[41] The method (specifically, according to any one of

[18] to

[40] ) wherein the growth of the animal cells is improved and / or the differentiation of the animal cells is promoted by culturing the animal cells in the presence of the iron ionophore.

[0009] According to the present invention, animal cells can be cultured.

[0010] Figure showing proliferation of bovine muscle stem cells in serum-free medium in the presence of hinokitiol. The vertical axis shows the fold increase. The horizontal axis shows group number. Figure showing proliferation of porcine preadipocytes (PSPA) in serum-free medium or in the presence of hinokitiol. The vertical axis shows the fold increase. The horizontal axis shows group number. Figure showing viable cell counts of HEK293 derivative cell lines subcultured in suspension culture in the presence of hinokitiol. Cell counts and subcultures were performed on days 3, 7, 10, 13, 17, and 20. The vertical axis shows cell number (x10 ６The horizontal axis indicates the number of days since the start of culture. This figure shows the proliferation of CD8-positive human T cells in serum-free medium in the presence of hinokitiol. The vertical axis indicates the fold increase. The horizontal axis indicates the group number.

[0011] <1> Active ingredient <1-1> Active ingredient In the present invention, an iron ionophore is used as the active ingredient.

[0012] The iron ionophore is also referred to as the "active ingredient."

[0013] The active ingredient can be used for culturing animal cells, specifically, animal cells can be cultured in the presence of the active ingredient.

[0014] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may improve the growth of animal cells, i.e., an effect of improving the growth of animal cells may be obtained. This effect is also referred to as a "growth-improving effect." In other words, the active ingredient may have the function of improving the growth of animal cells. This function is also referred to as a "growth-improving function." In other words, culturing animal cells in the presence of an active ingredient may improve the growth of animal cells compared to culturing animal cells in the absence of the active ingredient. Examples of culturing animal cells in the absence of an active ingredient include culturing animal cells under the same conditions as culturing animal cells in the presence of the active ingredient, except that the active ingredient is not used. The growth-improving effect may be obtained, for example, when animal cells are cultured in the substantial absence of serum (i.e., when animal cells are cultured in a medium substantially free of serum). The growth-improving effect may be obtained, for example, when animal cells are cultured in the substantial absence of animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of animal-derived albumin). The growth-enhancing effect may be obtained, for example, when animal cells are cultured in the substantial absence of serum and animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of serum and animal-derived albumin). The growth-enhancing effect may be obtained, for example, when the animal cells are MyoD+ cells (i.e., cells expressing MyoD). MyoD+ cells may be, for example, myoblasts. The growth enhancement may include an increase in proliferation rate. The growth-enhancing effect can be confirmed by measuring and comparing the growth (e.g., proliferation rate) of animal cells in the presence and absence of an active ingredient.

[0015] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells may be obtained. This effect is also referred to as a "differentiation-promoting effect." In other words, the active ingredient may have the function of promoting differentiation of animal cells. This function is also referred to as a "differentiation-promoting function." In other words, culturing animal cells in the presence of the active ingredient may promote differentiation of animal cells compared to culturing animal cells in the absence of the active ingredient. Examples of culturing animal cells in the absence of an active ingredient include culturing animal cells under the same conditions as culturing animal cells in the presence of the active ingredient, except that the active ingredient is not used. The differentiation-promoting effect may be obtained, for example, when animal cells are cultured in the substantial absence of serum (i.e., when animal cells are cultured in a medium substantially free of serum). The differentiation-promoting effect may be obtained, for example, when animal cells are cultured in the substantial absence of animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of animal-derived albumin). The differentiation-promoting effect may be obtained, for example, when animal cells are cultured in the substantial absence of serum and animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of serum and animal-derived albumin). The differentiation-promoting effect may be obtained, for example, when the animal cells are PAX7+ cells (i.e., cells expressing PAX7). PAX7+ cells may be, for example, satellite cells. Satellite cells may be activated. Examples of differentiation include myogenic differentiation. Examples of differentiation promotion include an increase in the differentiation rate and an increase in the proportion of differentiated cells. The differentiation-promoting effect can be confirmed by measuring and comparing the differentiation of animal cells (e.g., the differentiation rate and the proportion of differentiated cells) in the presence and absence of the active ingredient. Animal cell differentiation can be measured, for example, using the expression of a protein that serves as an indicator of differentiation.For example, during muscle differentiation, Sprouty1, Pax7, MyoD, Myogenin, and myosin heavy chain (MHC) can be expressed in sequence as differentiation progresses (Manuel Schmidt et al. Adult stem cells at work: regenerating skeletal muscle. Cell Mol Life Sci. 2019 Jul;76(13):2559-2570.).

[0016] By utilizing the active ingredient, specifically by culturing animal cells in the presence of the active ingredient, at least a growth-enhancing effect may be obtained.

[0017] By utilizing an active ingredient, specifically by culturing animal cells in the presence of the active ingredient, growth-enhancing effects and differentiation-promoting effects may be obtained.

[0018] The active ingredient may be used, for example, in place of transferrin. The active ingredient may, for example, substitute for the function of transferrin. The active ingredient may be less expensive than transferrin. Furthermore, transferrin is a protein and therefore requires refrigeration, whereas the active ingredient may be able to be stored at room temperature. Thus, the use of the active ingredient may be advantageous in terms of economy and / or storage compared to the use of transferrin.

[0019] In one aspect, a product may be produced by culturing animal cells. Examples of the product include cultured meat and target substances. The target substance is not particularly limited as long as it can be produced by animal cells. Examples of the target substance include proteins and viruses. Examples of the product include cultured meat. When a product is produced by culturing animal cells, the use of an active ingredient may improve product production. That is, by culturing animal cells in the presence of an active ingredient, product production may be improved compared to culturing animal cells in the absence of the active ingredient. For example, the use of an active ingredient may improve product production by improving the growth and / or promoting differentiation of animal cells. That is, one aspect of the growth-improving effect or differentiation-promoting effect may be an effect of improving product production. The effect of improving product production may be obtained, for example, when animal cells are cultured in conditions substantially free of serum (i.e., when animal cells are cultured in a medium substantially free of serum). The effect of improving product production may be obtained, for example, when animal cells are cultured under conditions substantially free of animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of animal-derived albumin). The effect of improving product production may be obtained, for example, when animal cells are cultured under conditions substantially free of serum and animal-derived albumin (i.e., when animal cells are cultured in a medium substantially free of serum and animal-derived albumin). Improvements in production include improvements in production amount and production rate.

[0020] The term "iron ionophore" may refer to a substance that has the function of increasing the permeability of iron ions through biological membranes. Specifically, the term "iron ionophore" may refer to, for example, a substance that binds to iron ions and passes through biological membranes. Examples of iron ionophores include hinokitiol (also known as "β-thujaplicin"), α-thujaplicin, and γ-thujaplicin. A particular example of an iron ionophore is hinokitiol. As the iron ionophore, one type of substance may be used, or two or more types of substances may be used in combination.

[0021] When an active ingredient can form a salt, the active ingredient may be used in its free form, its salt, or a combination thereof. That is, unless otherwise specified, the term "active ingredient" may refer to the active ingredient in its free form, its salt, or a combination thereof. "Free form" means a form in which no salt is formed. Also, when an active ingredient can form a hydrate, the active ingredient may be used in its non-hydrate form, its hydrate, or a combination thereof. That is, the term "active ingredient" (e.g., "active ingredient in its free form" or "salt of the active ingredient") may include both the non-hydrate and the hydrate unless otherwise specified. The active ingredient may be in any form, such as an ion, when used.

[0022] The salt is not particularly limited as long as it is acceptable for the purpose of the present invention. For example, when producing a product for consumption by culturing animal cells, an orally ingestible salt may be selected. For example, salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Specific examples of salts of basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid; and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. For example, examples of salts of succinic acid include sodium salts. One type of salt may be used, or two or more types of salts may be used in combination.

[0023] The active ingredient may be a commercially available product or may be obtained by appropriate manufacturing. The method for manufacturing the active ingredient is not particularly limited. The active ingredient can be manufactured, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The active ingredient may be purified to a desired degree or may not be purified. That is, the active ingredient may be a purified product, or a material containing the active ingredient. For example, the active ingredient may be a material containing the active ingredient at a content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0024] When a material containing an active ingredient is used, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material. When the active ingredient forms a salt or hydrate, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the mass of the salt or hydrate converted to the mass of an equimolar unhydrated free form.

[0025] <2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient (i.e., an iron ionophore).

[0026] The composition of the present invention can be used, for example, for culturing animal cells. That is, the composition of the present invention may be a composition for animal cell culture. The composition of the present invention can be used, for example, for culturing animal cells in the manner described in the method of the present invention described below.

[0027] The composition of the present invention (specifically, a composition for animal cell culture) may be, for example, a culture medium. Examples of culture media include basal medium, feed medium, and perfusion medium. "Basal medium" may refer to the medium used at the start of culture. Basal medium is also called "initial medium." "Fed-batch medium" may refer to the medium supplied to a culture system after the start of culture in fed-batch culture. "Perfusion medium" may refer to the medium supplied to a culture system after the start of culture in continuous culture (this is not limited to perfusion culture).

[0028] The composition of the present invention (specifically, a composition for animal cell culture) may be, for example, a medium additive. The term "medium additive" may refer to a composition that is added to a medium before use. Examples of media to which the medium additive is added include basal medium, feed medium, and perfusion medium.

[0029] Use of the composition of the present invention may improve the growth of animal cells, i.e., an effect of improving the growth of animal cells (growth-enhancing effect) may be obtained. For example, a growth-enhancing effect may be obtained by culturing animal cells in a medium containing the composition of the present invention. Furthermore, for example, a growth-enhancing effect may be obtained by culturing animal cells in a medium containing the composition of the present invention, which is a medium additive. In other words, the composition of the present invention may have a function of improving the growth of animal cells (growth-enhancing function). Thus, the composition of the present invention may be used to improve the growth of animal cells. That is, the composition of the present invention may be, for example, a composition for improving the growth of animal cells.

[0030] The use of the composition of the present invention may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells (differentiation-promoting effect) may be obtained. For example, a differentiation-promoting effect may be obtained by culturing animal cells in a medium containing the composition of the present invention. Furthermore, for example, a differentiation-promoting effect may be obtained by culturing animal cells in a medium containing the composition of the present invention, which is a medium additive. In other words, the composition of the present invention may have a function of promoting differentiation of animal cells (differentiation-promoting function). Thus, the composition of the present invention may be used to promote differentiation of animal cells. That is, the composition of the present invention may be, for example, a composition for promoting differentiation of animal cells.

[0031] Use of the composition of the present invention may result in at least a growth-enhancing effect.

[0032] The use of the composition of the present invention may provide a growth-enhancing effect and a differentiation-promoting effect. That is, the composition of the present invention may be, for example, a composition for enhancing the growth and promoting the differentiation of animal cells.

[0033] In one aspect, a product (e.g., cultured meat or a target substance) may be produced by culturing animal cells. When a product is produced by culturing animal cells, the use of the composition of the present invention may improve production of the product. For example, the use of the composition of the present invention may improve the growth and / or promote differentiation of the animal cells, thereby improving production of the product. In other words, one aspect of the composition of the present invention (specifically, a composition for animal cell culture) may be a composition for improving production of a product.

[0034] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. Ingredients other than the active ingredient are also referred to as "additional ingredients." That is, the active ingredient may be used as is or in combination with additional ingredients in the composition of the present invention. The additional ingredients may be a single ingredient, or two or more ingredients may be used in combination.

[0035] That is, the present invention provides a method for producing the composition of the present invention, which comprises using an active ingredient. The manner of use of the active ingredient in the production of the composition of the present invention is not particularly limited, as long as a composition containing the active ingredient is obtained. Use of the active ingredient in the production of the composition of the present invention includes blending the active ingredient into the composition. "Bundling an active ingredient into a composition" may mean containing the active ingredient in the composition. "Bundling an active ingredient into a composition" may specifically mean combining the active ingredient with an additional ingredient.

[0036] The additional component is not particularly limited as long as it can achieve the object of the present invention (for example, a growth-improving effect or a differentiation-promoting effect can be obtained). The additional component can be appropriately selected depending on various conditions, such as the type of animal cell and the mode of use of the composition of the present invention. Examples of the additional component include medium components. Medium components will be described later. For example, at least when the composition of the present invention is a medium, the composition of the present invention may contain medium components. Specific examples of the additional component include iron. That is, the composition of the present invention may contain, for example, iron.

[0037] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. That is, the additional components include additives used in formulation. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and surfactants. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0038] The form of the composition of the present invention is not particularly limited, and the composition of the present invention may be in any form, such as powder, flakes, tablets, paste, or liquid.

[0039] The content and content ratio of each component (i.e., active component and optional additional component) in the composition of the present invention are not particularly limited as long as the object of the present invention can be achieved (e.g., a growth-enhancing effect or a differentiation-promoting effect can be obtained). That is, for example, the content of the active component in the composition of the present invention may be an effective amount (e.g., an amount that can achieve a growth-enhancing effect or a differentiation-promoting effect). The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions, such as the type of animal cell and the mode of use of the composition of the present invention.

[0040] The content of the active ingredient in the composition of the present invention is more than 0% (w / w) and not more than 100% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) or more, 10 ppb (w / w) or more, 100 ppb (w / w) or more, 1 ppm (w / w) or more, 10 ppm (w / w) or more, 100 ppm (w / w) or more, 1000 ppm (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more, and may be 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 1000 ppm (w / w) or less, 100 The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) to 1 ppm (w / w), 1 ppm (w / w) to 10 ppm (w / w), 10 ppm (w / w) to 100 ppm (w / w), 100 ppm (w / w) to 1000 ppm (w / w), 1000 ppm (w / w) to 1% (w / w), 1% (w / w) to 10% (w / w), or 10% (w / w) to 20% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 1 ppb (w / w) to 10% (w / w), 1 ppb (w / w) to 1% (w / w), or 1 ppb (w / w) to 1000 ppm (w / w). When the composition of the present invention contains two or more active ingredients, the contents of those two or more active ingredients in the composition of the present invention may be set independently or in total within the range of the content of the active ingredients in the composition of the present invention exemplified above (with the proviso that the total content of those two or more active ingredients in the composition of the present invention is 100% (w / w) or less).In addition, when the composition of the present invention contains two or more active ingredients, the "content of the active ingredients in the composition of the present invention" means the total content of those two or more active ingredients in the composition of the present invention, unless otherwise specified.

[0041] In one embodiment, the composition of the present invention may be substantially free of serum. "The composition of the present invention is substantially free of serum" may mean that the serum content in the composition of the present invention is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include a case where the serum content in the composition of the present invention is 0 (zero) (i.e., the composition of the present invention does not contain serum).

[0042] In one embodiment, the composition of the present invention may be substantially free of animal-derived albumin. "The composition of the present invention is substantially free of animal-derived albumin" may mean that the content of animal-derived albumin in the composition of the present invention is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include a case where the content of animal-derived albumin in the composition of the present invention is 0 (zero) (i.e., the composition of the present invention does not contain animal-derived albumin).

[0043] In one embodiment, the composition of the present invention may be substantially free of transferrin. "The composition of the present invention is substantially free of transferrin" may mean that the content of transferrin in the composition of the present invention is 0.001% (w / w) or less, 0.0001% (w / w) or less, 0.00001% (w / w) or less, or 0.000001% (w / w) or less, and may also include a case where the content of transferrin in the composition of the present invention is 0 (zero) (i.e., the composition of the present invention does not contain transferrin).

[0044] The content of each component (i.e., the active component and any additional component) in the composition of the present invention can be set, for example, so as to obtain the concentration of each component in the medium in the method of the present invention described below.

[0045] When the composition of the present invention contains two or more components, the components may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination. For example, the composition of the present invention may be provided as a set consisting of a package of an active ingredient and a package of an additional ingredient. In such a case, the components contained in the set can be used together as appropriate when used.

[0046] <3> Method of the Present Invention The method of the present invention is a method for culturing animal cells, which comprises a step of culturing animal cells in the presence of an active ingredient (i.e., an iron ionophore). This step is also referred to as a "step of culturing animal cells."

[0047] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may improve the growth of the animal cells, i.e., an effect of improving the growth of the animal cells (growth-improving effect) may be obtained. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be, for example, a method for improving the growth of animal cells.

[0048] The use of an active ingredient, specifically, culturing animal cells in the presence of the active ingredient, may promote differentiation of animal cells, i.e., an effect of promoting differentiation of animal cells (differentiation-promoting effect) may be obtained. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be, for example, a method for promoting differentiation of animal cells.

[0049] By utilizing the active ingredient, specifically by culturing animal cells in the presence of the active ingredient, at least a growth-enhancing effect may be obtained.

[0050] The use of an active ingredient, specifically by culturing animal cells in the presence of the active ingredient, may provide a growth-enhancing effect and a differentiation-promoting effect. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be, for example, a method for improving the growth of animal cells and promoting their differentiation.

[0051] The purpose of culturing animal cells is not particularly limited.

[0052] In one aspect, a product (e.g., cultured meat or a target substance) may be produced by culturing animal cells. That is, if animal cells are capable of forming cultured meat through culture, then cultured meat can be produced by culturing those cells. Cultured meat may be composed primarily of muscle tissue. Thus, "animal cells are capable of forming cultured meat through culture" may mean, for example, that animal cells are capable of differentiating through culture to form muscle tissue, and specifically, that animal cells are capable of differentiating into myotube cells through culture to form muscle tissue. Furthermore, if animal cells have the ability to produce a target substance, then the target substance can be produced by culturing those cells. That is, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be a method for producing a product, which includes a step of culturing animal cells in the presence of an active ingredient. Furthermore, the step of culturing animal cells may specifically be a step of culturing animal cells in the presence of an active ingredient to produce a product. In the method for producing cultured meat, cultured animal cells may form cultured meat. In the method for producing a target substance, cultured animal cells may produce the target substance. In other words, if animal cells can produce a target substance by culturing, the target substance can be produced by culturing the cells. Examples of target substances include proteins and viruses. When the target substance includes a virus, the method for producing the target substance may further include a step of causing the animal cells to produce the virus. When a product is produced by culturing animal cells, the production of the product may be improved by using an active ingredient. For example, the production of the product may be improved by improving the growth and / or promoting differentiation of the animal cells through the use of an active ingredient. In other words, one aspect of the method of the present invention (specifically, the method for culturing animal cells) may be a method for improving the production of a product.

[0053] "Animal cells" refers to cells of an animal. The animal cells are not particularly limited. The animal cells can be selected appropriately depending on various conditions, such as the purpose of culturing the animal cells. For example, when producing cultured meat by culturing animal cells, the animal cells are not particularly limited, as long as they can form cultured meat through culture. "Animal" may refer to organisms classified under the kingdom Animalia. Examples of animals include vertebrates and aquatic organisms. Examples of vertebrates include mammals, birds, reptiles, amphibians, and fish. Examples of vertebrates in particular include mammals, birds, and fish. Examples of vertebrates in particular include mammals. Examples of mammals include primates such as humans, monkeys, and chimpanzees; rodents such as hamsters, mice, rats, and guinea pigs; other terrestrial mammals such as cows, pigs, sheep, goats, rabbits, horses, deer, water buffalo, reindeer, donkeys, camels, dogs, and cats; and aquatic mammals such as whales, dolphins, reindeer, and sea lions. Birds include chickens, turkeys, ducks, geese, guinea fowl, quail, and ostriches. Fish include eels, tuna, grouper, sea bream, salmon, cod, and pufferfish. Aquatic organisms include aquatic mammals and fish, as well as crustaceans such as shrimp and crab, shellfish such as scallops and oysters, and other aquatic organisms such as squid and octopus. Aquatic organisms particularly include fish and crustaceans. Animals suitable for producing cultured meat include animals raised for meat. Meat animals include those animals listed above whose meat can be used for food. Meat animals (particularly mammals) particularly include cattle, pigs, sheep, goats, and rabbits. Meat animals (particularly mammals) particularly include cattle and pigs. Meat animals (particularly mammals) particularly include cattle. Examples of meat-producing animals (particularly birds) include chickens, turkeys, ducks, geese, guinea fowl, quails, and ostriches. Examples of meat-producing animals (particularly birds) include chickens, more particularly. Examples of meat-producing animals (particularly aquatic organisms) include the fish and crustaceans exemplified above. Examples of meat-producing animals (particularly aquatic organisms) include eels, tuna, and shrimp, more particularly. The tissues or cells from which animal cells are derived are not particularly limited.Examples of tissues or cells from which animal cells are derived include ovaries, kidneys, adrenal glands, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, melanocytes, skin, spleen, liver, lungs, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testes, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eyes, brain, and nervous tissue. Animal cells may or may not be differentiated. Specific examples of animal cells include germ cells, somatic cells, stem cells, and progenitor cells. Germ cells include sperm and eggs. Somatic cells include myoblasts, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells (e.g., keratinocytes, corneocytes, etc.), endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nerve cells, glial cells, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells, skeletal muscle cells), pancreatic beta cells, melanocytes, and mononuclear cells. Stem cells include adult stem cells such as hematopoietic stem cells, satellite cells (muscle stem cells), neural stem cells, mesenchymal stem cells, mammary stem cells, olfactory mucosa stem cells, neural crest stem cells, hepatic stem cells, pancreatic stem cells, germ stem cells, intestinal stem cells, and hair follicle stem cells; pluripotent stem cells such as embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, and induced pluripotent stem cells (iPS cells); and cancer stem cells. Progenitor cells include satellite cells, pancreatic progenitor cells, vascular progenitor cells, vascular endothelial progenitor cells, and hematopoietic progenitor cells (such as CD34-positive cells derived from umbilical cord blood). Animal cells suitable for producing cultured meat include stem cells such as satellite cells (muscle stem cells), mesenchymal stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), as well as myoblasts. Animal cells suitable for producing cultured meat include satellite cells (muscle stem cells) and myoblasts, in particular. Myoblasts may be classified as stem cells.

[0054] "Animal cells satisfy a certain condition" means that the animal cells must satisfy the condition for at least a portion of the culture period, and does not require the animal cells to satisfy the condition for the entire culture period. In other words, "animal cells satisfy a certain condition" is not limited to cases where animal cells satisfy the condition at the start of culture (i.e., culture is initiated using animal cells that satisfy the condition), but may also include cases where animal cells satisfy the condition after the start of culture. For example, "animal cells are myoblasts" is not limited to cases where culture is initiated using animal cells that are myoblasts, but may also include cases where animal cells differentiate into myoblasts after the start of culture.

[0055] The animal cells are cultured in the presence of an active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the animal cells are cultured in a medium containing the active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the active ingredient is supplied to the culture system during the culture of the animal cells.

[0056] The active ingredient may be used in the form of, for example, the composition of the present invention for culturing animal cells. The composition of the present invention may be used, for example, as a culture medium for culturing animal cells. That is, for example, when the composition of the present invention is a culture medium, animal cells may be cultured in the composition of the present invention (i.e., the culture medium). That is, "culturing animal cells in a culture medium containing an active ingredient" may also include culturing animal cells in the composition of the present invention, which is a culture medium. Furthermore, the composition of the present invention may be used, for example, as a culture medium additive for culturing animal cells. That is, for example, when the composition of the present invention is a culture medium additive, the composition of the present invention (i.e., the culture medium additive) may be added to the culture medium, and animal cells may be cultured in the culture medium supplemented with the composition of the present invention. That is, "culturing animal cells in a culture medium containing an active ingredient" may also include culturing animal cells in a culture medium supplemented with the composition of the present invention, which is a culture medium additive. Furthermore, for example, the composition of the present invention or a culture medium supplemented with the composition of the present invention may be supplied to a culture system during animal cell culture. That is, "supplying an active ingredient to a culture system during animal cell culture" may also include supplying the composition of the present invention or a culture medium supplemented with the composition of the present invention to a culture system during animal cell culture.

[0057] The medium composition and culture conditions are not particularly limited as long as the purpose of animal cell culture is achieved, provided that the animal cell culture is carried out in the presence of an active ingredient. The medium composition and culture conditions can be set appropriately depending on various conditions, such as the type of animal cell and the purpose of animal cell culture. For example, when producing cultured meat by culturing animal cells, the medium composition and culture conditions are not particularly limited as long as cultured meat can be formed by culturing the animal cells. Except for the presence of an active ingredient, the animal cell culture can be carried out using, for example, a conventional medium and conventional conditions used for animal cell culture, either as is or with appropriate modifications. When producing cultured meat by culturing animal cells, except for the presence of an active ingredient, the animal cell culture can be carried out using, for example, a conventional medium and conventional conditions used for the formation of muscle tissue by animal cell differentiation (e.g., the production of cultured meat), either as is or with appropriate modifications.

[0058] Culture can be carried out using, for example, a liquid medium. Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Continuous culture includes perfusion culture and chemostat culture. The medium at the start of culture is also called the "initial medium" or "basal medium." The medium supplied to a culture system (e.g., initial medium) in fed-batch culture is also called the "feed medium." The medium supplied to a culture system (e.g., initial medium) in continuous culture (not limited to perfusion culture) is also called the "perfusion medium." Supplying a feed medium or perfusion medium to a culture system in fed-batch or continuous culture is also simply called "medium supply." Medium supply may be carried out throughout the entire culture period or only for a portion of the culture period. Medium supply may be carried out continuously or intermittently. During culture (particularly continuous culture such as perfusion culture), the culture medium may be withdrawn. The culture medium may be withdrawn throughout the entire culture period, or only for a portion of the culture period. The culture medium may be withdrawn continuously or intermittently. The culture medium may be withdrawn and the medium may be supplied simultaneously, or not. The culture vessel may be coated with a cell adhesion molecule such as fibronectin. Culturing may be carried out three-dimensionally, for example, using a scaffold. When producing cultured meat by culturing animal cells, culturing may be carried out three-dimensionally, for example, using a scaffold that matches the shape of the cultured meat to be formed.

[0059] The media used for culture, for example, the basal medium, the feed medium, and the perfusion medium, can be selected independently.

[0060] The medium used for the culture may be a commercially available medium or an appropriately prepared medium.

[0061] The medium used for the culture includes a medium containing components essential for culturing animal cells (for example, a carbon source, a nitrogen source, inorganic salts, etc.).

[0062] Specific examples of media used for culture include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0063] Furthermore, specific examples of media used for culture (for example, media particularly used for culturing stem cells (particularly pluripotent stem cells)) include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Examples of suitable medium include hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0064] Other commercially available media include animal cell culture media such as CELLiST Basal Media BASAL3 and BASAL10 (Ajinomoto Co., Inc.), Opti-MEM (Thermo Fisher Scientific), RPMI 1640 (Thermo Fisher Scientific), CD293 (Thermo Fisher Scientific), CHO-S-SFMII (Thermo Fisher Scientific), CHO-SF (Sigma-Aldrich), EX-CELL CD CHO (Sigma-Aldrich), EX-CELL™302 (Sigma-Aldrich), IS CHO-CD (Irvine Scientific), and IS CHO-CDXP (Irvine Scientific).

[0065] The media exemplified above may be used for culture by adding, for example, an active ingredient.

[0066] Furthermore, the medium used for culture may be, for example, the composition of the present invention (specifically, the composition of the present invention that is a medium). That is, when the composition of the present invention is a medium, the composition of the present invention may be used for culture as is, or may be appropriately prepared as a liquid medium of a desired composition. For example, the composition of the present invention may be diluted with an aqueous medium such as water or an aqueous buffer solution to prepare a liquid medium and use it for culture.

[0067] Furthermore, the medium used for culture may be, for example, a medium supplemented with the composition of the present invention (specifically, the composition of the present invention as a medium additive). The medium supplemented with the composition of the present invention may be a commercially available medium or an appropriately prepared medium.

[0068] The medium may contain various medium components. Examples of medium components include carbon sources, amino acid sources, peptides, proteins, vitamins, fatty acids, lipids, organic acids, nucleic acids, amines, antioxidants, inorganic components, pH buffers, growth factors, cytokines, hormones, cell adhesion factors, extracellular matrix components, serum, antibiotics, and gene expression inducers. Any of these medium components may be essential or effective for, for example, the survival or proliferation of animal cells. Any of these medium components may be pre-contained in the medium exemplified above, or may be added to the medium exemplified above.

[0069] Examples of carbon sources include sugars such as glucose, fructose, sucrose, and maltose.

[0070] The amino acid source may include amino acids. In addition, both peptides and proteins may be examples of amino acid sources. The amino acids may include glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, proline, and ornithine. The amino acids may be, for example, L-form.

[0071] Examples of peptides include dipeptides and tripeptides. Specific examples of peptides include glycylglycylglycine and soybean peptide. The descriptions of amino acids also apply mutatis mutandis to the amino acids that make up peptides.

[0072] Examples of proteins include albumin and transferrin.

[0073] Vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, and vitamin K, and precursors thereof.

[0074] Fatty acids include oleic acid, arachidonic acid, and linoleic acid.

[0075] Lipids include cholesterol.

[0076] Examples of organic acids include pyruvic acid (such as sodium pyruvate).

[0077] Nucleic acids include hypoxanthine and thymidine.

[0078] The amines include polyamines such as putrescine.

[0079] Antioxidants include lipoic acid.

[0080] Inorganic components include sodium, potassium, calcium, magnesium, phosphorus, and various trace elements (e.g., Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn). Specific examples of inorganic components include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate. Iron, in particular, can be iron (II), iron (III), iron (III), or a mixture thereof. Iron salts include iron sulfate (e.g., iron(II) sulfate), iron nitrate (e.g., iron(III) nitrate and iron(II) nitrate), iron chloride (e.g., iron(III) chloride and iron(II) chloride), iron sulfide (e.g., iron(III) sulfide and iron(II) sulfide), and ammonium iron citrate (e.g., ammonium iron(III) citrate). Iron (e.g., iron salts) can be, for example, hydrates. As the iron, one kind of component may be used, or two or more kinds of components may be used in combination.

[0081] Examples of pH buffers include sodium bicarbonate, phosphate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine).

[0082] Examples of growth factors include fibroblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), activin A, and insulin-like growth factor-1 (IGF-1). Examples of FGF include basic fibroblast growth factor (bFGF). Examples of growth factors include bFGF in particular. That is, at least bFGF may be used as a medium component. That is, the medium may contain at least bFGF. Examples of bFGF include bovine basic fibroblast growth factor (bbFGF). "Bovine basic fibroblast growth factor (bbFGF)" may refer to bovine-derived bFGF. The term "bovine" in reference to bbFGF may refer to organisms of the genus Bos. Bos organisms include Bos taurus. bbFGF is not limited to bFGF found in organisms of the genus Bos, and may be a modified form thereof.

[0083] Cytokines include interleukins.

[0084] Hormones include dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin.

[0085] Examples of cell adhesion factors or extracellular matrix components include Type I collagen, Type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine.

[0086] Antibiotics include amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin.

[0087] In one embodiment, the medium may be substantially free of serum. For example, the medium may be substantially free of serum throughout the entire culture period. "The medium is substantially free of serum" may mean that the serum content in the medium is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include a case where the serum content in the medium is 0 (zero) (i.e., the medium does not contain serum).

[0088] In one aspect, the medium may be substantially free of animal-derived albumin. Examples of animals from which albumin is derived include the animals exemplified for animal cells. The medium may be substantially free of animal-derived albumin, for example, throughout the entire culture period. "The medium is substantially free of animal-derived albumin" may mean that the content of animal-derived albumin in the medium is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include a case where the content of animal-derived albumin in the medium is 0 (zero) (i.e., the medium does not contain animal-derived albumin).

[0089] Various components, such as active ingredients, may be contained in the initial medium, feed medium, perfusion medium, or a combination thereof. That is, various components, such as active ingredients, may be supplied to the medium alone or in any combination during the culture process. These components may be supplied once, multiple times, or continuously. The compositions (e.g., the types and / or concentrations of the components) of the initial medium, feed medium, and perfusion medium may or may not be the same. That is, the types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium or perfusion medium. Furthermore, the concentrations of the components contained in the initial medium may or may not be the same as the concentrations of the components contained in the feed medium or perfusion medium. For example, when a feed medium is used for perfusion culture, the compositions of the initial medium and the feed medium may be the same. Furthermore, two or more feed media or perfusion media with different compositions (e.g., the types and / or concentrations of the components contained) may be used. For example, when the feed medium or perfusion medium is supplied intermittently multiple times, the composition of the feed medium or perfusion medium may or may not be the same for each supply. In addition, various components such as active ingredients may be supplied to the medium in a form not contained in the feed medium or perfusion medium, such as a powder.

[0090] The seeding amount of animal cells at the start of culture is, for example, 1 × 10 in terms of viable cell number. 2 cells / mL or more, 1×10 3 cells / mL or more, 1×10 4 cells / mL or more, 1×10 5 cells / mL or more, 1×10 6 cells / mL or more, or 1×10 7 cells / mL or more, but not more than 1 x 10 8 cells / mL or less, 1×10 7 cells / mL or less, 1×10 6 cells / mL or less, 1×10 5 cells / mL or less, 1×10 4 cells / mL or less, or 1 x 10 3The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL or less in terms of the number of viable cells. 2 ~1×10 3 cells / mL, 1 × 10 3 ~1×10 4 cells / mL, 1 × 10 4 ~1×10 5 cells / mL, 1 × 10 5 ~1×10 6 cells / mL, 1 × 10 6 ~1×10 7 cells / mL, or 1 x 10 7 ~1×10 8 The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL in terms of the number of viable cells. 2 ~1×10 8 cells / mL, 1 × 10 3 ~1×10 7 cells / mL, or 1 x 10 3 ~1×10 6 The number of viable cells may be measured using, for example, a viable cell autoanalyzer, Vi-CELL TM Measurement can be performed using XR (Beckman Coulter).

[0091] The culture may be carried out under, for example, 5 to 15% CO ２ CO etc. ２The cultivation may be carried out under a pH-containing atmosphere. The pH of the medium may be, for example, near neutral. "Near neutral" may mean, for example, pH 6 to 8, pH 6.5 to 7.5, or pH 6.8 to 7.2. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The cultivation temperature may be, for example, 30 to 38°C. The culture period may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, 15 days or more, or 20 days or more, or 60 days or less, 50 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less, or any compatible combination thereof. Specific examples of the culture period may be, for example, 1 to 60 days, 3 to 25 days, or 5 to 20 days. Culture may be continued, for example, until the product is produced to a desired level.

[0092] The amount (e.g., concentration in the medium) and ratio of amounts used (e.g., concentration ratio in the medium) of each component (i.e., active ingredient and optional additional ingredient) in the method of the present invention are not particularly limited as long as the object of the present invention can be achieved (e.g., a growth-enhancing effect or a differentiation-promoting effect can be obtained). That is, for example, the amount of the active ingredient used in the method of the present invention may be an effective amount (e.g., an amount that can achieve a growth-enhancing effect or a differentiation-promoting effect). Specifically, for example, the concentration of the active ingredient in the medium may be an effective concentration (e.g., a concentration that can achieve a growth-enhancing effect or a differentiation-promoting effect). The amount and ratio of amounts used of each component in the method of the present invention can be appropriately determined depending on various conditions, such as the type of animal cell and the mode of use of the composition of the present invention.

[0093] The concentration of the active ingredient in the medium is, for example, 1 nM or more, 3 nM or more, 5 nM or more, 10 nM or more, 30 nM or more, 50 nM or more, 100 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, 600 nM or more, 700 nM or more, 1 μM or more, 1.5 μM or more, 2 μM or more, 2.5 μM or more, 3 μM or more, 5 7 μM or more, 10 μM or more, 15 μM or more, 20 μM or more, 25 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, or 70 μM or more, 100 μM or less, 70 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 25 μM or less, 20 μM or less, 15 μM or less, 10 μM or less, 7 μM or less, 5 μM or less, 3 The concentration may be 0.1 μM or less, 2.5 μM or less, 2 μM or less, 1.5 μM or less, 1 μM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, or 3 nM or less, or any compatible combination thereof. Specifically, the concentration of the active ingredient in the medium is, for example, 1 nM to 3 nM, 3 nM to 5 nM, 5 nM to 10 nM, 10 nM to 30 nM, 30 nM to 50 nM, 50 nM to 100 nM, 100 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM. nM, 600 nM to 700 nM, 700 nM to 1 μM, 1 μM to 1.5 μM, 1.5 μM to 2 μM, 2 μM to 2.5 μM, 2.5 μM to 3 μM, 3 μM to 5 μM, 5 μM to 7 μM, 7 μM to 10 μM, 10 μM to 15 μM, 15 μM to 20 μM, 20 μM to 25 μM, 25 μM to 30 The concentration of the active ingredient in the medium may be, for example, 1 nM to 100 μM, 5 nM to 30 μM, 10 nM to 10 μM, or 10 nM to 1 μM.In one embodiment, the concentration of the active ingredient in the medium may be, specifically, 2.5 μM or less, 2 μM or less, 1.5 μM or less, 1 μM or less, 700 nM or less, 600 nM or less, 500 nM or less, or 400 nM or less. For example, when at least hinokitiol is selected as the active ingredient, the concentration of hinokitiol or the total concentration of the active ingredients including hinokitiol in the medium may be, specifically, 2.5 μM or less, 2 μM or less, 1.5 μM or less, 1 μM or less, 700 nM or less, 600 nM or less, 500 nM or less, or 400 nM or less. In one embodiment, the concentration of the active ingredient in the medium may be, specifically, for example, 1 nM to 2.5 μM, 3 nM to 2 μM, 5 nM to 1.5 μM, or 10 nM to 1 μM. For example, when at least hinokitiol is selected as the active ingredient, the concentration of hinokitiol or the total concentration of the active ingredients including hinokitiol in the medium may be 1 nM to 2.5 μM, 3 nM to 2 μM, 5 nM to 1.5 μM, or 10 nM to 1 μM. When the medium contains two or more active ingredients, the concentrations of those two or more active ingredients in the medium may be set independently or in total within the range of the concentrations of the active ingredients in the medium exemplified above. When the medium contains two or more active ingredients, the "concentration of the active ingredients in the medium" refers to the total concentration of those two or more active ingredients in the medium, unless otherwise specified.

[0094] The animal cells may be cultured in the presence of iron. "Culturing animal cells in the presence of a certain component (e.g., iron)" may mean, for example, that the animal cells are cultured in a medium containing the component. "Culturing animal cells in the presence of a certain component (e.g., iron)" may mean, for example, that the component is supplied to a culture system when the animal cells are cultured.

[0095] When the medium contains iron, the concentration of iron in the medium may be, for example, 200 nM or more, 500 nM or more, 1 μM or more, 1.5 μM or more, 2 μM or more, 3 μM or more, 5 μM or more, 7 μM or more, 10 μM or more, 15 μM or more, 20 μM or more, 25 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, 70 μM or more, 100 μM or more, 150 μM or more, or 200 μM or more, 500 μM or more, 200 μM or more, 150 μM or more, 100 μM or more, 70 μM or more, 50 μM or more, 40 μM or more, 30 μM or more, 25 μM or more, 20 μM or more, 15 μM or more, 10 μM or more, 7 μM or more, 5 μM or more, 3 μM or more, 2 μM or more, 1.5 μM or more, 1 μM or more, or 500 The iron concentration in the medium may be, for example, 200 nM to 500 nM, 500 nM to 1 μM, 1 μM to 1.5 μM, 1.5 μM to 2 μM, 2 μM to 3 μM, 3 μM to 5 μM, 5 μM to 7 μM, 7 μM to 10 μM, 10 μM to 15 μM, 15 μM to 20 μM, 20 μM to 25 μM, 25 μM to 30 μM, 30 μM to 40 μM, 40 μM to 50 μM, 50 μM to 70 μM, 70 μM to 100 μM, 100 μM to 150 μM, 150 μM to 200 μM, or 200 μM to 500 μM. The iron concentration in the medium may be, for example, 200 nM to 500 μM, 500 nM to 100 μM, 1 μM to 50 μM, or 1.5 μM to 30 μM. When the medium contains two or more forms of iron, the concentrations of those two or more forms of iron in the medium, either independently or in total, may be set within the range of iron concentrations in the medium exemplified above. When the medium contains two or more forms of iron, the "iron concentration in the medium" refers to the total concentration of those two or more forms of iron in the medium, unless otherwise specified. The molar concentration of iron is calculated based on the number of moles of iron constituting the compound. For example, in the case of 1 μM iron(III) sulfide (Fe2S3), the molar concentration of iron is 2 μM.

[0096] Each of the various components, such as the active ingredient, may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component" means that the component is contained in the medium during at least a portion of the culture period, and does not necessarily have to be contained in the medium throughout the entire culture period. For example, the active ingredient and iron may coexist in the medium during at least a portion of the culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, or may be supplied to the medium after the start of culture. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, and further supplied to the medium after the start of culture (e.g., after the active ingredient has been consumed).

[0097] Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above throughout the entire culture period, or only during a portion of the culture period. That is, "culture is carried out in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" means that the concentration of the component in the medium is within the range for at least a portion of the culture period, but does not necessarily need to be within the range for the entire culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above, for example, at the start of culture, or may be supplied to the medium after the start of culture to achieve the concentrations exemplified above. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above at the start of culture, and then further supplied to the medium after the start of culture (e.g., after the component has been consumed) to achieve the concentrations exemplified above.

[0098] The length of the "partial culture period" is not particularly limited as long as the object of the present invention can be achieved (e.g., a growth-improving effect or a differentiation-promoting effect can be obtained). The length of the "partial culture period" can be appropriately set depending on various conditions, such as the type of animal cell and the length of the culture period. The "partial culture period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the total culture period. Furthermore, the "partial culture period" may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, or 15 days or more.

[0099] Furthermore, the concentrations of various components, such as active ingredients, in the culture medium may be set to the concentrations exemplified above, for example, as average values ​​over a specific period during culture. That is, "culture is performed in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" may mean that the average concentration of the component in the medium over a specific period during culture falls within the range of concentrations. The "average concentration of a certain component in the medium over a specific period during culture" is not particularly limited as long as it allows for understanding fluctuations in the concentration of the component over a specific period during culture. For example, it may mean the average concentration of the component in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes over a specific period during culture. The "specific period during culture" may refer to the entire culture period or a portion of the culture period. The "partial culture period" is as described above.

[0100] Each of the various components, such as the active ingredient, may be supplied to the medium throughout the entire culture period, or may be supplied to the medium only during a portion of the culture period. The "part of the culture period" is as described above. Each of the various components, such as the active ingredient, may be supplied to the medium continuously or intermittently, for example. Each of the various components, such as the active ingredient, may be supplied to the medium every day, or every few days, for example.

[0101] The concentrations of various components, such as active ingredients, in the feed medium or perfusion medium may be within the range of the concentrations of the components in the media exemplified above, or may be, for example, 1-fold or more, 1.1-fold or more, 1.3-fold or more, 1.5-fold or more, 2-fold or more, 3-fold or more, 5-fold or more, 7-fold or more, 10-fold or more, 15-fold or more, or 20-fold or more, or 100-fold or less, 70-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 7-fold or less, 5-fold or less, 3-fold or less, 2-fold or less, or any combination thereof that is consistent therewith. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 1 to 2 times, 1.1 to 2 times, 1.3 to 2 times, 1.5 to 2 times, 2 to 3 times, 3 to 5 times, 5 to 7 times, 7 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 30 times, 20 to 50 times, 20 to 70 times, or 20 to 100 times the concentrations of the components in the above-exemplified media. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 1 to 100 times, 2 to 50 times, or 5 to 20 times the concentrations of the components in the above-exemplified media.

[0102] The concentrations of any of the components, including the active ingredient, can be measured by known methods used to detect or identify compounds, such as HPLC, UPLC, LC / MS, GC / MS, and NMR.

[0103] Animal cells can be cultured as described above. In one embodiment, a product (e.g., cultured meat or a target substance) may be obtained by culturing animal cells as described above. When a target substance is produced, the target substance may accumulate in the culture, for example, in the medium, on the cell surface, inside the cells, or a combination thereof. The target substance may be recovered from the culture as appropriate. That is, the method for producing a target substance may further include a step of recovering the target substance.

[0104] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the applications exemplified above. That is, the present invention discloses, for example, the use of the active ingredient for culturing animal cells, the use of the active ingredient for improving the growth of animal cells, the use of the active ingredient for promoting the differentiation of animal cells, and the use of the active ingredient in the production of the composition of the present invention, such as a composition for animal cell culture.

[0105] The present invention also discloses active ingredients for use in the above-mentioned applications, i.e., active ingredients for use in culturing animal cells, active ingredients for use in improving the growth of animal cells, active ingredients for use in promoting the differentiation of animal cells, and active ingredients for use in producing a composition for animal cell culture.

[0106] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0107] Example 1: Evaluation of the activity of hinokitiol using bovine muscle stem cells in serum-free medium (1-1) Preparation of bovine muscle stem cells Muscle tissue was collected from edible beef shank, and CD56-positive cells were isolated using a magnetically activated cell sorting system (MACS) to prepare bovine muscle stem cells.

[0108] (1-2) Evaluation of Hinokitiol Activity Using Bovine Muscle Stem Cells in Serum-Free Medium Bovine muscle stem cells obtained in (1-1) were suspended in serum-free medium. The serum-free medium consisted of Advanced DMEM (Thermo Fisher Scientific, Cat No. 12491015) supplemented with 20% FBS (Thermo Fisher Scientific, Cat No. 10270106), 10% horse serum (Thermo Fisher Scientific, Cat No. 16050-130), and 1% GlutaMAX. TM Supplement (Thermo Fisher Scientific, Cat No. 35050061) was added to the cell suspension at 1 μg / cm 2Cells were seeded onto fibronectin (Corning, Cat#F2006)-coated 12-well plates (BD FALCON, Cat#353046) at a cell density of 25,000 cells / 0.2 mL, and the medium was changed every other day. On day 4 of culture, the cells were passaged into serum-free medium containing hinokitiol, serum-free medium containing hinokitiol and iron, serum-free medium containing iron, serum-free medium containing transferrin and iron, serum-free medium containing transferrin, or serum-free medium without hinokitiol or transferrin, and the medium was changed every other day. All media were based on E8 medium (DMEM / F12, 64 mg / L l-ascorbic acid 2-phosphate magnesium, 14 μg / L sodium selenium, 19.4 mg / L insulin, 543 mg / L NaHCO3, and 2 μg / L TGFβ1 adjusted to pH 7.4 and 340 mOsm; Chen, G. et al. Nature Methods 8, 424-429 (2011)) supplemented with 5 ng / mL HGF (Peprotech, Cat. #294HGN005), 20 ng / mL EGF (Sigma, Cat. #E9644), and 10 ng / mL bFGF (Peprotech, Cat. #AF-450-62). E8 medium contains approximately 1.63 μM iron. Table 1 summarizes the group numbers and media. Cell counts and passages were performed every 4 days, and proliferation rates were calculated for two consecutive passages from day 4 to day 12.

[0109] The results are shown in Figure 1. Hinokitiol was found to promote cell proliferation in bovine muscle stem cells. This indicates that iron ionophores such as hinokitiol are effective in culturing animal cells (especially in promoting proliferation). It was also revealed that iron ionophores such as hinokitiol can replace transferrin. Furthermore, increasing the iron concentration sometimes further improved the cell proliferation promoting effect of hinokitiol on bovine muscle stem cells.

[0110]

[0111] Example 2: Evaluation of activity of hinokitiol using porcine preadipocytes (PSPA) in serum-free medium (2-1) Obtaining PSPA As porcine preadipocytes (PSPA), a preadipocyte cell line derived from fetal pig subcutaneous tissue, as described in Nakajima, et al. Biochem Biophys Res Commun. 2003 Sep 26;309(3):702-8, was obtained from RIKEN RCB and used.

[0112] (2-2) Evaluation of Hinokitiol Activity Using Porcine Preadipocytes (PSPA) in Serum-Free Medium The porcine preadipocytes (PSPA) described in (2-1) were cultured in serum-free medium. The serum-free medium consisted of DMEM (Gibco, Cat No. 10313-021) supplemented with 10% FBS (Thermo Fisher Scientific, Cat No. 10270106) and 1% GlutaMAX. TM The serum-free medium used was a medium based on the E8 medium. A 12-well plate was cultured at 10,000 cells / cm. 2 PSPA were seeded at 100°C, and the cells were passaged in serum medium, serum-free medium containing transferrin, and serum-free medium containing hinokitiol and iron (without transferrin), with the medium changed every other day. Table 2 summarizes the group numbers, media, and additives. Cell counts were performed 4 days after the start of culture, and the fold increase in cell number was calculated.

[0113] The results are shown in Figure 2. Hinokitiol was found to have a cell proliferation promoting effect on porcine preadipocytes.

[0114]

[0115] Example 3: Confirmation of proliferation of HEK293 cells with the addition of hinokitiol using a suspension culture system (3-1) Obtaining HEK293 cells Viral Production Cells 2.0 (Thermo Fisher Scientific: A49784), a clone cell line derived from the HEK293 cell line, was used as HEK293. The HEK293 cell line is widely known as a cell line derived from human fetal kidney. (3-2) Evaluation of the activity of hinokitiol using HEK293 cells in a suspension culture system GlutaMAX 1000 mg was added to 30 mL Viral Production Medium (Thermo Fisher Scientific: A4817901) in a 125 mL Erlenmeyer flask (VIOLAMO, SEF125V). TM Viral Production Cells 2.0, a clone cell line derived from HEK293 cells, was seeded in medium supplemented with Supplement (Thermo Fisher Scientific, Cat No. 35050061) to a final concentration of 4 mM, and cultured in an incubator at 37°C and 8% CO for two passages. Then, 30 mL of Basal 10 (Ajinomoto Healthy Supply Co., Ltd., CELLiST TM The cells were subcultured in a Basal media (BASAL10)-based medium or in our company's medium supplemented with hinokitiol at a final concentration of 500 nM. The number of viable cells was measured every 3 or 4 days using a Vi-CELL BLU viable cell autoanalyzer (Becman) and then subcultured repeatedly for a total of 20 days.

[0116] The results are shown in Figure 3. The addition of hinokitiol was found to promote cell proliferation of HEK293 cells.

[0117] Example 4: Evaluation of activity of hinokitiol using CD8-positive human T cells in serum-free medium (4-1) Evaluation of activity of hinokitiol using CD8-positive human T cells in serum-free medium CD3 monoclonal antibody (Thermo Fisher Scientific, Cat No. 16-0037-85) was mixed with PBS and added to a 24-well plate. After incubation at 37°C for 1 hour, the plate was washed three times with PBS. CTS was used as the base medium. TM OpTmizer TM T Cell Expansion (Thermo Fisher Scientific, Cat No. A104851), CTS TM The medium used was supplemented with Immune Cell SR (Thermo Fisher Scientific, Cat. No. A2596101), 200 mM L-glutamine (Thermo Fisher Scientific, Cat. No. 25030081), P / S / Amphotericin B (Fujifilm-Wako, Cat. No. 161-23181), and IL2 (PeproTech, Cat. No. 200-02). In a coated 24-well plate, hinokitiol-supplemented media groups were prepared as shown in Table 3, and CD8+ T cells (LONZA, Cat. No. 2W-300) were cultured in each group. After 7 days, cell counts were performed and proliferation rates were calculated.

[0118]

[0119] The results are shown in Figure 4. Hinokitiol was found to have a cell proliferation-promoting effect on CD8-positive human T cells.

[0120] According to the present invention, animal cells can be cultured.

Claims

1. A composition for animal cell culture containing an iron ionophore.

2. The composition according to claim 1, which is a composition for improving the growth and / or promoting the differentiation of animal cells.

3. The composition according to claim 1 or 2, which is a culture medium or a culture medium additive.

4. The composition of claim 3, wherein the medium is a basal medium, a feed medium, or a perfusion medium.

5. The composition according to claim 1 or 2, wherein the iron ionophore is at least one component selected from the group consisting of hinokitiol, α-thujaplicin, and γ-thujaplicin.

6. The composition of claim 1 or 2, wherein the iron ionophore is hinokitiol.

7. The composition according to claim 1 or 2, further comprising iron.

8. The composition of claim 7, wherein the iron is an iron salt.

9. The composition of claim 1 or 2, wherein the animal is a mammal, a bird, a fish, or a crustacean.

10. The composition according to claim 1 or 2, wherein the animal is a meat animal.

11. The composition according to claim 1 or 2, wherein the animal is a cow, a pig or a chicken.

12. The composition of claim 1 or 2, wherein the animal is a human.

13. The composition of claim 1 or 2, wherein the cells are muscle stem cells, adipose precursor cells, kidney-derived cells, or T cells.

14. The composition of claim 1 or 2, which is substantially free of serum.

15. The composition of claim 1 or 2, which is substantially free of animal-derived albumin.

16. The composition of claim 1 or 2, which is substantially free of transferrin.

17. The composition according to claim 1 or 2, which has the function of improving the growth of animal cells and / or the function of promoting the differentiation of animal cells.

18. A method for producing a product, comprising culturing animal cells in the presence of an iron ionophore.

19. The method of claim 17, wherein the product is cultured meat.

20. A method for culturing animal cells, comprising culturing the animal cells in the presence of an iron ionophore.

21. A method for improving the growth of animal cells, comprising culturing the animal cells in the presence of an iron ionophore.

22. A method for promoting differentiation of animal cells, comprising culturing the animal cells in the presence of an iron ionophore.

23. The method of any one of claims 18 to 22, wherein the iron ionophore is at least one member selected from the group consisting of hinokitiol, α-thujaplicin, and γ-thujaplicin.

24. The method of any one of claims 18 to 22, wherein the iron ionophore is hinokitiol.

25. The method of any one of claims 18 to 22, wherein the culturing is carried out in the presence of iron.

26. The method of claim 25, wherein the iron is an iron salt.

27. The method according to any one of claims 18 to 22, wherein the concentration of the iron ionophore in the medium during the culture is 1 nM to 100 μM.

28. The method according to any one of claims 18 to 22, wherein the concentration of the iron ionophore in the medium during the culture is 10 nM to 10 μM.

29. The method according to claim 24, wherein the concentration of hinokitiol in the medium during the culture is 1 nM to 2.5 μM.

30. The method according to claim 24, wherein the concentration of hinokitiol in the medium during the culture is 10 nM to 1 μM.

31. The method according to claim 25, wherein the concentration of iron in the medium during the culture is 200 nM to 500 μM.

32. The method of any one of claims 18 to 22, wherein the animal is a mammal, a bird, a fish, or a crustacean.

33. The method of any one of claims 18 to 22, wherein the animal is a meat animal.

34. The method of any one of claims 18 to 22, wherein the animal is a cow, a pig, or a chicken.

35. The method of any one of claims 18, 20, 21, and 22, wherein the animal is a human.

36. The method of any one of claims 18 to 22, wherein the cells are muscle stem cells or preadipocytes.

37. The method of any one of claims 18, 20, 21, and 22, wherein the cell is a kidney-derived cell or a T cell.

38. The method according to any one of claims 18 to 22, wherein the culturing is carried out in a medium substantially free of serum.

39. The method according to any one of claims 18 to 22, wherein the culturing is carried out in a medium substantially free of animal-derived albumin.

40. The method according to any one of claims 18 to 22, wherein the culturing is carried out in a medium substantially free of transferrin.

41. The method according to any one of claims 18 to 22, wherein the growth of animal cells is improved and / or the differentiation of animal cells is promoted by culturing the animal cells in the presence of the iron ionophore.

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