Composition for basal medium for culturing animal cells

Abstract

The present invention relates to a composition for a basal medium for culturing animal cells. This composition for a basal medium for culturing animal cells contains a yeast extract, an amino acid, an inorganic salt, and a sugar, and the amino acid in the composition contains cystine and / or cysteine, and glutamine.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for a basal medium for animal cell culture and use thereof, a method for culturing animal cells, and the like. BACKGROUND

[0002] Cell culture refers to proliferation / maintenance of cells isolated from tissues of living organisms in a culture solution. In recent years, development of cultured meat, and in particular, research on culture of animal cells outside of living organisms is being promoted. A culture medium for culturing animal cells is generally composed of inorganic salts, carbohydrates, amino acids, vitamins, proteins, peptides fatty acids, lipids, serum, and the like.

[0003] "Cell agriculture" refers to a technology in which cells collected from animals and plants are proliferated by directly providing nutrients to the cells, and the cells are produced as food. Meat, fish, and the like produced using the cell agriculture technology are collectively referred to as "cell-based food". In cell-based food, a substance equivalent to "meat" is sometimes referred to as "cultured meat".

[0004] From the viewpoints of ensuring food safety and reducing environmental burden, cell-based food is being developed worldwide. For commercialization of cell-based food, two items, raw materials / manufacturing processes suitable for food production, and reduction of manufacturing costs, are particularly important. IntegriCulture Co., Ltd. (Bunkyo-ku, Tokyo, Japan) has successfully developed I-MEM 1.0, which reproduces a composition for a basal medium, which is the most important element in the production of cell-based food, using only food additive raw materials that are recognized as food (Press Release PRTIMES "IntegriCulture, independently developed basal medium composition "I-MEM" consisting entirely of food raw materials "I-MEM" began accepting orders worldwide on October 26 (Wednesday); https: / / prtimes.jp / main / html / rd / p / 000000025.000034252.html).

[0005] I-MEM 1.0 has achieved foodization of the components of the culture medium and reduction of manufacturing costs, which are major issues in the production of cell-cultured meat and the like. However, with regard to reduction of manufacturing costs, even I-MEM 1.0 cannot be said to be sufficient, and development of a more inexpensive and safe composition for a basal medium is required.

[0006] Japanese Patent Application Publication No. 2005-532057 describes a culture medium for culturing cells that does not contain animal-derived proteins. Claims 1 and 2 of Japanese Patent Application Publication No. 2005-532057 are as follows.

[0007] 1. A cell culture medium free of animal protein, comprising a soybean hydrolysate and a yeast hydrolysate.

[0008] 2. The cell culture medium free of animal protein according to claim 1, wherein the soybean hydrolysate is present at a concentration of at least 0.05% (w / v) and the yeast hydrolysate is present at a concentration of at least 0.05% (w / v).

[0009] As understood from the description according to claims 1 and 2, the medium of Japanese Patent Application Publication No. 2005-532057 has soybean hydrolysate as an essential component. In addition, the main purpose of the culture of cells in Japanese Patent Application Publication No. 2005-532057 is to produce biological products such as viruses, recombinant proteins, and the like in the cells (for example, claims 23, paragraphs

[0011] ,

[0012] , and the like of Japanese Patent Application Publication No. 2005-532057).

[0010] It is desirable to develop a medium for animal cell culture that can be used as a food product, can suppress the use of food additives and the like while maintaining culturability, and has a small environmental burden.

[0011] Prior Art Documents

[0012] Patent Documents

[0013] Patent Document 1: Japanese Patent Application Publication No. 2005-532057

[0014] Non-Patent Documents

[0015] Non-Patent Document 1: Press Release PRTIMES "IntegriCulture, Independently Developed Basic Medium Entirely Composed of Food Raw Materials, I-MEM (アイメム), Begins Accepting Orders in the World on October 26 (Wednesday)" https: / / prtimes.jp / main / html / rd / p / 000000025.000034252.html

[0016] Summary of the Invention

[0017] Problems to be Solved by the Invention

[0018] The present inventors and others conducted intensive research in order to solve the above problems, and as a result, found that by replacing a part of the composition of Dulbecco's Modified Eagle Medium (DMEM medium) with yeast extract, and further adding cystine and / or cysteine, and glutamine, it is possible to maintain the same culturability as the conventional DMEM medium while suppressing the addition of amino acids as food additives, thereby completing the present invention.

[0019] Method of Solving the Problems

[0020] The present invention includes the following methods, but is not limited thereto.

[0021] I [1]

[0023] A composition for a basal culture medium for animal cell culture, comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids in the composition include cystine and / or cysteine, and glutamine. [2]

[0025] According to the composition for basal culture medium for animal cell culture described in [1], when the composition for basal culture medium is set to 100, the content of all amino acids in the composition is 0.002 to 1.0 parts by mass. [3]

[0027] The composition for basal culture medium for animal cell culture according to [1] or [2], wherein the proportions of cystine and / or cysteine ​​and glutamine are 50% or more relative to all amino acids in the above composition. [4]

[0029] The composition for basal culture medium for animal cell culture according to any one of [1] to [3], wherein when the composition for basal culture medium is set to 100, the content of cystine and / or cysteine ​​and glutamine in the composition is 0.001 to 1.0 parts by weight. [5]

[0031] The composition for basal culture medium for animal cell culture according to any one of [1] to [4], wherein the content of cystine and / or cysteine ​​and glutamine in the composition is 0.1 to 5.0 parts by weight per unit solid component of the composition for basal culture medium. [6]

[0033] The composition for basal culture medium for animal cell culture according to any one of [1] to [5], wherein when the composition for basal culture medium is set to 100, the content of cystine and / or cysteine ​​in the composition is 0.0001 to 0.02 parts by mass. [7]

[0035] The composition for basal culture medium for animal cell culture according to any one of [1] to [6], wherein the content of cystine and / or cysteine ​​in the composition is 0.005 to 1.0 parts by weight per unit solid component of the composition for basal culture medium. [8]

[0037] The composition for basal culture medium for animal cell culture according to any one of [1] to [7], wherein when the composition for basal culture medium is set to 100, the content of glutamine in the composition is 0.001 to 1.0 parts by mass. [9]

[0039] The composition for basal culture medium for animal cell culture according to any one of [1] to [8], wherein the content of glutamine in the composition is 0.1 to 5.0 parts by weight per unit solid component of the composition for basal culture medium.

[10]

[0041] The composition for basal culture medium for animal cell culture according to any one of [1] to [9], wherein the yeast extract is a high-amino acid type yeast extract.

[11]

[0043] The composition for basal culture medium for animal cell culture according to any one of [1] to

[10] , wherein when the composition for basal culture medium is set to 100, the total amino acid content of the yeast extract is 0.75 parts by mass or more.

[12]

[0045] The composition for basal culture medium for animal cell culture according to any one of [1] to

[11] , wherein the yeast extract is a low-nucleic acid yeast extract.

[13]

[0047] The composition for basal culture medium for animal cell culture according to any one of [1] to

[12] , wherein when the composition for basal culture medium is set to 100, the total nucleic acid content of the yeast extract is 2.0 parts by mass or less.

[14]

[0049] The composition for basal culture medium for animal cell culture according to any one of [1] to

[13] , wherein the yeast extract is a yeast extract with high amino acid content and low nucleic acid content.

[15]

[0051] The composition for basal culture medium for animal cell culture according to any one of [1] to

[14] , wherein when the composition for basal culture medium is set to 100, the content of the yeast extract is 0.05 to 20.0 parts by mass.

[16]

[0053] The composition for basal culture medium for animal cell culture according to [1] or [2] further comprises vitamins.

[17]

[0055] A method for culturing animal cells, the method comprising: culturing animal cells in a basal culture medium for animal cells, said basal culture medium for animal cells comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids are cystine and / or cysteine, and glutamine.

[18]

[0057] According to the cultivation method described in

[17] , the yeast extract is an extract of yeast with high amino acid content, yeast with low nucleic acid content, or yeast with both high amino acid content and low nucleic acid content.

[19]

[0059] An animal cell obtained by culturing using the culture method described in

[17] or

[18] .

[20]

[0061] A processed food containing the animal cells described in

[19] .

[0062] II [1]

[0064] A basic culture medium for animal cells, comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids are cystine and glutamine. [2]

[0066] According to the basic culture medium for animal cells described in [1], when the basic culture medium is set to 100, the content of the above amino acids is 0.005~1.0 parts by mass. [3]

[0068] According to the animal cell basal culture medium described in [1] or [2], the proportion of cystine and glutamine in the above-mentioned amino acids is more than 50%. [4]

[0070] According to the basic culture medium for animal cells described in [1] or [2], when the basic culture medium is set to 100, the content of cystine and glutamine in the above amino acids is 0.0025~1.0 parts by mass. [5]

[0072] The basal culture medium for animal cells according to [1] or [2], wherein the yeast extract is a high-amino acid type yeast extract. [6]

[0074] According to the animal cell basal culture medium described in [1] or [2], when the basal culture medium is set to 100, the total amino acid content of the above yeast extract is 0.75 parts by mass or more. [7]

[0076] The basal culture medium for animal cells as described in [1] or [2], wherein the yeast extract is a low-nucleic acid yeast extract. [8]

[0078] According to the animal cell basal culture medium described in [1] or [2], when the basal culture medium is set to 100, the total nucleic acid content of the above yeast extract is less than 2.0 parts by mass. [9]

[0080] The basal culture medium for animal cells according to [1] or [2], wherein the yeast extract is a yeast extract with high amino acid content and low nucleic acid content.

[10]

[0082] According to the basic culture medium for animal cells described in [1] or [2], when the basic culture medium is set to 100, the content of the above yeast extract is 0.05 to 20.0 parts by mass.

[11]

[0084] The basal culture medium for animal cells as described in [1] or [2] further contains vitamins.

[12]

[0086] The basal culture medium for animal cells according to [1] or [2] contains calcium chloride as the aforementioned inorganic salt.

[13]

[0088] A method for culturing animal cells, the method comprising: culturing animal cells in a basal culture medium for animal cells, said basal culture medium for animal cells comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids are cystine and glutamine.

[14]

[0090] According to the cultivation method of

[13] , the above yeast extract is an extract of yeast with a high amino acid content.

[15]

[0092] According to the culture method in

[13] , the yeast extract is an extract of a low-nucleic acid yeast.

[16]

[0094] According to the culture method of

[13] , the above yeast extract is an extract of yeast with high amino acid type and low nucleic acid type.

[17]

[0096] An animal cell obtained by culturing using the culture method described in

[13] .

[18]

[0098] A processed food containing the animal cells described in

[17] .

[19]

[0100] Use of yeast extract in a basal culture medium for animal cells, which is composed of amino acids, inorganic salts and sugars, wherein the amino acids are cystine and glutamine.

[0101] The effects of the invention

[0102] Yeast extract is a flavoring agent containing amino acids, peptides, and nucleic acids found in yeast. This invention, by replacing a portion of the culture medium composition with yeast extract, reduces the amount of ingredients added to the composition compared to conventional commercially available basal culture medium compositions, thereby reducing the cost and environmental burden required for animal cell culture. Attached Figure Description

[0103] [ Figure 1 ] Figure 1 The results of cell images from a study showing the minimum types of amino acids required to be added in Example 1 are shown.

[0104] [ Figure 2 ] Figure 2 The results of cell images from a culture experiment using MDBK and HDFa cells in Example 2 are shown.

[0105] [ Figure 3 ] Figure 3 The results of comparing viable cell counts by absorbance measurement at 450 nm are shown in Example 4, using various yeast extracts. From left to right, the results for each passage are: DMEM, PC (positive control), yeast extract A, yeast extract B, yeast extract C, yeast extract D, and NC (negative control).

[0106] [ Figure 4 ] Figure 4 The results of comparing viable cell counts by absorbance measurement at 450 nm are shown in Example 5, using yeast extracts A and B, with the yeast extract concentration increased to 2.0 g / L, and calcium chloride added to the basal medium. In each passage, from left to right, are the results for PC (positive control), yeast extract A, yeast extract B, and NC (negative control).

[0107] [ Figure 5 ] Figure 5The results of counting viable cells at the third passage time point are shown in Example 6 when cells from various organs derived from chickens were cultured using yeast extract A and yeast extract AB. From left to right, the results for liver, muscle, lung, kidney, and brain are shown in PC (positive control), yeast extract A, and yeast extract AB.

[0108] [ Figure 6 ] Figure 6 The results of investigating cell counts in RL34 cells (rats with normal liver cells) cultured using yeast extract A in Example 7 are shown. The vertical axis represents the cell count value when cultured in DMEM medium as a positive control, set to 1.0. The horizontal axis P1, P2, and P3 represent the results of passage 1, passage 2, and passage 3, respectively.

[0109] [ Figure 7 ] Figure 7 The results of investigating cell counts in MDBK cells (bovine normal kidney-derived cells) cultured using yeast extract A in Example 7 are shown. The vertical axis represents the cell count value when cultured in DMEM medium as a positive control, set to 1.0. The horizontal axis P1, P2, and P3 represent the results of passage 1, passage 2, and passage 3, respectively.

[0110] [ Figure 8 ] Figure 8 The results of a cell count survey in Example 7, where bovine muscle-derived cells were cultured using yeast extract A, are shown. The vertical axis represents the cell count value when DMEM medium, used as a positive control, was set to 1.0. The horizontal axis, P1, P2, and P3, represent the results for the 1st, 2nd, and 3rd generations, respectively.

[0111] [ Figure 9 ] Figure 9 The results are from three passages of MDBK cells in basal culture media with various amino acid additions, as described in Example 8. Figure 9 The vertical axis represents the number of living cells.

[0112] [Figure 10] Figure 10 is an example of using the inclusion L-cysteine or L-cysteine ​​basal medium for MDBK cells (cell line) Figure 10A ), RL34 cells (cell line) Figure 10B ), duck liver-derived cells (primary culture cells) Figure 10C The results of three passage cultures are shown in Figure 10. The vertical axis of Figure 10 represents the number of viable cells after three passage cultures. Detailed Implementation

[0113] This invention includes, without limitation, the following methods. Unless otherwise stated, the technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed in this specification are merely illustrative and not intended to be limiting. In this specification, the reference to "in one method" is not intended to limit the scope of the invention.

[0114] 1. A composition for basal culture medium used in animal cell culture.

[0115] In one embodiment, the present invention relates to a composition for a basal culture medium used in animal cell culture.

[0116] The above-mentioned composition for basal culture medium for animal cell culture comprises yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids in the composition include cystine and / or cysteine, and glutamine. In one embodiment, the above-mentioned composition for basal culture medium for animal cell culture is composed of yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids are cystine and / or cysteine, and glutamine.

[0117] The content of all amino acids in the above-mentioned composition for basal culture medium for animal cell culture is not particularly limited. Here, "all amino acids" refers to the constituent components of the composition for basal culture medium for animal cell culture, specifically amino acids added to the composition in the form of amino acid monomers, and is distinct from the amino acids contained in the yeast extract described later. "All amino acids" includes, in addition to cysteine ​​and / or cysteine, and glutamine, which are essential components of the above composition, other types of amino acids added to the composition for basal culture medium for animal cell culture.

[0118] When the composition for basal culture medium is set to 100, the content of all amino acids in the above composition is, without limitation, 5.0 parts by weight or less, 3.0 parts by weight or less, 2.0 parts by weight or less, 1.0 parts by weight or less, 0.5 parts by weight or less, 0.1 parts by weight or less, 0.08 parts by weight or less, and 0.07 parts by weight or less. When the composition for basal culture medium is set to 100, the content of all amino acids in the above composition is, without limitation, 0.0005 parts by weight or more, 0.001 parts by weight or more, 0.002 parts by weight or more, 0.003 parts by weight or more, 0.004 parts by weight or more, 0.005 parts by weight or more, 0.01 parts by weight or more, 0.03 parts by weight or more, 0.05 parts by weight or more, and 0.06 parts by weight or more.

[0119] When the composition for basal culture medium is set to 100, the content of all amino acids in the above composition is non-limitingly 0.0005~5.0 parts by weight, 0.001~5.0 parts by weight, 0.001~3.0 parts by weight, 0.002~3.0 parts by weight, 0.002~2.0 parts by weight, 0.002~1.0 parts by weight, 0.003~2.0 parts by weight, 0.005~1.0 parts by weight, 0.01~0.5 parts by weight, 0.03~0.1 parts by weight, 0.002~0.07 parts by weight, or 0.004~0.07 parts by weight. When the composition for basal culture medium is set to 100, the content of the above amino acids is preferably 0.002~1.0 parts by weight.

[0120] The ratio of cystine and / or cysteine, and glutamine is not particularly limited. Cystine and / or cysteine, and glutamine may be in equal amounts, or each may be in greater amounts. Preferably, glutamine is in greater amounts. In one embodiment, glutamine is at least 1.5 times, 2 times, 3 times, 5 times, 8 times, or 10 times the amount of cystine and / or cysteine ​​by mass. In another embodiment, glutamine is approximately 12 times the amount of cystine and / or cysteine.

[0121] Cystine, cysteine, and glutamine can be in the L-form or the D-form. Cystine, cysteine, and glutamine can be partially in the L-form and partially in the D-form. In one configuration, cystine, cysteine, and glutamine are in the L-form.

[0122] "The amino acids in the above composition include cystine and / or cysteine, and glutamine" means that the main components of the above amino acids are cystine and / or cysteine, and glutamine. In one embodiment, the proportion of cystine and / or cysteine, and glutamine relative to all amino acids in the above composition is 50% or more. In another embodiment, the proportion of cystine and / or cysteine, and glutamine relative to all amino acids in the above composition is 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more. In one embodiment, the proportion of cystine and / or cysteine, and glutamine relative to all amino acids in the above composition is 100%. However, it is not limited to "the proportion of cystine and / or cysteine, and glutamine is 100%". It can be speculated that when more other amino acids are added to the above composition, the nutritional value is improved, and the proliferative capacity may be enhanced.

[0123] When the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine ​​and glutamine in the above composition is, without limitation, 0.0005 parts by mass or more, 0.0008 parts by mass or more, 0.001 parts by mass or more, 0.002 parts by mass or more, 0.0025 parts by mass or more, 0.003 parts by mass or more, 0.005 parts by mass or more, 0.01 parts by mass or more, 0.03 parts by mass or more, 0.05 parts by mass or more, or 0.06 parts by mass or more.

[0124] When the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine, and glutamine in the above composition is not limited to 0.00025~5.0 parts by weight, 0.0005~5.0 parts by weight, 0.0008~5.0 parts by weight, 0.0008~3.0 parts by weight, 0.001~5.0 parts by weight, 0.001~3.0 parts by weight, 0. 0.001~2.0 parts by weight, 0.001~1.0 parts by weight, 0.002~5.0 parts by weight, 0.002~3.0 parts by weight, 0.002~1.0 parts by weight, 0.0025~1.0 parts by weight, 0.003~2.0 parts by weight, 0.005~1.0 parts by weight, 0.01~0.5 parts by weight, 0.03~0.1 parts by weight, 0.002~0.07 parts by weight. In one embodiment, when the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine, and glutamine in the amino acids of the above composition is 0.001~1.0 parts by weight. In another embodiment, when the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine, and glutamine in the amino acids of the above composition is 0.002~1.0 parts by weight.

[0125] Based on the amount of solid components per unit of the composition for basal culture medium, the content of cystine and / or cysteine, and glutamine in the above composition is not limited to 0.025~25.0 parts by weight, 0.05~25.0 parts by weight, 0.08~25.0 parts by weight, 0.08~15.0 parts by weight, 0.1~25.0 parts by weight, 0.1~15.0 parts by weight, 0.1~10.0 parts by weight, 0.1~5.0 parts by weight, 0.2~25.0 parts by weight, 0.2~15 parts by weight, 0.2~5.0 parts by weight, 0.25~5.0 parts by weight, 0.3~10.0 parts by weight, 0.5~5.0 parts by weight, 0.3~2.5 parts by weight, 0.4~2.0 parts by weight, and 0.1~3.5 parts by weight. In one embodiment, the content of cystine and / or cysteine, and glutamine in the composition is 0.1 to 5.0 parts by weight per unit solid component of the composition for basal culture medium. In another embodiment, the content of cystine and / or cysteine, and glutamine in the composition is 0.2 to 5.0 parts by weight per unit solid component of the composition for basal culture medium.

[0126] When the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine ​​in the above composition is not limited to 0.00001~0.2 parts by mass, 0.00005~0.15 parts by mass, 0.00008~0.1 parts by mass, 0.0001~0.1 parts by mass, 0.0001~0.08 parts by mass, 0.0001~0.04 parts by mass, 0.0001~0.02 parts by mass, 0.0001~0.01 parts by mass, 0.0002~0.1 parts by mass, 0.0002~0.08 parts by mass, 0.0002~0.05 parts by mass, 0.0002~0.02 parts by mass, 0.0002~0.01 parts by mass, 0.0002~0.005 parts by mass, or 0.0004~0.008 parts by mass. In one embodiment, when the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine ​​in the composition is 0.0001 to 0.02 parts by mass.

[0127] Based on the amount of solid components per unit of the composition for basal culture medium, the content of cystine and / or cysteine ​​in the above composition is, without limitation, 0.0005~10.0 parts by weight, 0.0025~7.5 parts by weight, 0.004~5.0 parts by weight, 0.005~5.0 parts by weight, 0.005~4.0 parts by weight, 0.005~2.0 parts by weight, 0.005~1.0 parts by weight, 0.005~0.5 parts by weight, 0.01~5.0 parts by weight, 0.01~4.0 parts by weight, 0.01~2.5 parts by weight, 0.01~1.0 parts by weight, 0.01~0.5 parts by weight, 0.01~0.30 parts by weight, or 0.02~0.40 parts by weight. In one embodiment, the content of cystine and / or cysteine ​​in the composition is 0.005 to 1.0 parts by weight, based on the amount of solid components per unit of the composition for basal culture medium.

[0128] When the composition for the basal culture medium is set to 100, the content of glutamine in the amino acids in the above composition is not limited to 0.00025~5.0 parts by weight, 0.0005~5.0 parts by weight, 0.0008 parts by weight~5.0 parts by weight, 0.0008 parts by weight~3.0 parts by weight, 0.001~5.0 parts by weight, 0.001~3.0 parts by weight, 0.001~2.0 parts by weight, 0.001~1.0 parts by weight, 0.002~5.0 parts by weight, 0.002~3.0 parts by weight, 0.002~1.0 parts by weight, 0.0025~1.0 parts by weight, 0.003~2.0 parts by weight, 0.005~1.0 parts by weight, 0.01~0.5 parts by weight, 0.03~0.1 parts by weight, and 0.002~0.06 parts by weight. In one embodiment, when the composition for the basal culture medium is set to 100, the content of glutamine in the amino acids of the above composition is 0.001 to 1.0 parts by weight. In another embodiment, when the basal culture medium is set to 100, the content of glutamine in the amino acids of the above composition is 0.002 to 1.0 parts by weight.

[0129] Based on the amount of solid components per unit of the composition for basal culture medium, the content of glutamine in the above composition is not limited to 0.025~25.0 parts by weight, 0.05~25.0 parts by weight, 0.08~25.0 parts by weight, 0.08~15.0 parts by weight, 0.1~25.0 parts by weight, 0.1~15.0 parts by weight, 0.1~10.0 parts by weight, 0.1~5.0 parts by weight, 0.2~25.0 parts by weight, 0.2~15 parts by weight, 0.2~5.0 parts by weight, 0.25~5.0 parts by weight, 0.3~10.0 parts by weight, 0.5~5.0 parts by weight, 0.3~2.5 parts by weight, 0.4~2.0 parts by weight, and 0.1~3.2 parts by weight. In one embodiment, the glutamine content in the composition is 0.1 to 5.0 parts by weight per unit solid component of the composition for basal culture medium. In another embodiment, the glutamine content in the composition is 0.2 to 5.0 parts by weight per unit solid component of the composition for basal culture medium.

[0130] The amino acids contained in the above-mentioned basal culture medium composition for animal cell culture are cystine and / or cysteine, and glutamine, preferably excluding amino acids other than cystine and / or cysteine, and glutamine. "Amino acids other than cystine and / or cysteine, and glutamine" is not particularly limited and includes both natural and non-natural amino acids. As an example, amino acids other than cystine and / or cysteine, and glutamine include: isoleucine, leucine, threonine, tryptophan, valine, histidine, phenylalanine, methionine, lysine (including lysine hydrochloride), tyrosine, arginine, glycine, and serine.

[0131] Yeast extract (also known as yeast flavor enhancer) is a substance extracted from yeast cells. Typically, as the main component, it contains proteins and related substances such as peptides, amino acids, and nucleic acids obtained from their breakdown, as well as minerals and vitamins. It is known to be used as a seasoning, livestock feed, health supplement, culture medium for microorganisms, and quality improver for processed foods.

[0132] The amino acids contained in the yeast extract are components of the composition for basal culture medium used for animal cell culture, and are distinct from the amino acids (i.e., cystine and / or cysteine, and glutamine) added to the composition for basal culture medium used for animal cell culture in the form of amino acid monomers.

[0133] The type of yeast cells derived from the yeast extract included in the above-mentioned composition for the basal culture medium for animal cell culture is not particularly limited. In one embodiment (i.e., without limitation), examples of yeast cells include baker's yeast, brewer's yeast, sake yeast, and round yeast. One embodiment includes baker's yeast (scientific name: Saccharomyces cerevisiae).

[0134] Yeast extracts are known to include, based on their composition, high-amino acid type yeast extracts, high-nucleic acid / high-amino acid type yeast extracts, and high-nucleic acid type yeast extracts. Yeast extracts rich in specific amino acids, organic acids, and / or nucleic acids are also known. For example, particularly high-glutamic acid type yeast extracts containing high concentrations of glutamic acid, high-succinic acid type yeast extracts containing high concentrations of succinic acid, and high-nucleic acid type yeast extracts containing high concentrations of inosinic acid and guanylic acid.

[0135] The yeast extract described above is not limited to a high-amino acid type yeast extract. The yeast extract described above is not limited to a low-nucleic acid type yeast extract. The yeast extract described above is not limited to a high-amino acid and low-nucleic acid type yeast extract.

[0136] In one embodiment, the high-amino acid yeast extract contains more than 30,000 mg, more than 35,000 mg, more than 40,000 mg, or more than 45,000 mg of total amino acids per 100g of yeast extract.

[0137] When each 100g of yeast extract contains 30,000mg of total amino acids, the amino acid content in the yeast extract is 30%. In Example 3 described later, the yeast extract was added in such a way that the final concentration in the culture medium reached 0.25g / L to 2.0g / L. When the amino acid content in the yeast extract was 30%, the amino acid concentration in the culture medium was 0.075g / L to 0.6g / L (1000g). In this case, when the composition for the basal culture medium was set to 100, the total amino acid content of the yeast extract was 0.75 parts by weight to 6.0 parts by weight.

[0138] When the composition for the basal culture medium is set to 100, the total amino acid content of the yeast extract is, non-limitingly, 0.30 parts by mass or more, 0.50 parts by mass or more, 0.60 parts by mass or more, 0.75 parts by mass or more, 1.0 parts by mass or more, or 1.5 parts by mass or more. In one embodiment, when the basal culture medium is set to 100, the total amino acid content of the yeast extract is 0.75 parts by mass or more.

[0139] When the composition for the basal culture medium is set to 100, the total amino acid content of the above yeast extract is not limited to 20 parts by mass or less, 15 parts by mass or less, 10 parts by mass or less, 8 parts by mass or less, or 6 parts by mass or less.

[0140] When the composition for the basal culture medium is set to 100, the total amino acid content of the yeast extract is, without limitation, 0.30 parts by mass or more and 20 parts by mass or less, 0.50 parts by mass or more and 10 parts by mass or less, 0.60 parts by mass or more and 8 parts by mass or less, or 0.75 parts by mass or more and 6 parts by mass or less.

[0141] In one manner, in yeast extracts, low-nucleic acid yeast extracts contain less than 10%, less than 8%, less than 5%, less than 3%, and less than 2% nucleic acids.

[0142] In Example 3 described later, yeast extract was added to achieve a final concentration of 0.25 g / L to 2.0 g / L in the culture medium. With a nucleic acid content of 10% in the yeast extract, the nucleic acid concentration in the culture medium was 0.025 g / L to 0.2 g / L (1000 g). In this case, when the composition for the basal culture medium was set to 100, the total nucleic acid content of the yeast extract was 0.25 parts by weight to 2.0 parts by weight.

[0143] When the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is, non-limitingly, 3.0 parts by weight or less, 2.0 parts by weight or less, 1.5 parts by weight or less, 1.0 parts by weight or less, 0.7 parts by weight or less, 0.5 parts by weight or less, or 0.3 parts by weight or less. In one embodiment, when the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is 2.0 parts by weight or less.

[0144] When the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is, non-limitingly, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.10 parts by mass or more, 0.20 parts by mass or more, or 0.25 parts by mass or more. In one embodiment, when the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is 0.25 parts by mass or more.

[0145] When the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is, without limitation, 0.05 parts by mass or more and 2.0 parts by mass or less, 0.10 parts by mass or more and 2.0 parts by mass or less, 0.20 parts by mass or more and 2.0 parts by mass or less, or 0.25 parts by mass or more and 2.0 parts by mass or less.

[0146] The yeast extract contained in the above-mentioned composition of the basic culture medium for animal cell culture may be one or more.

[0147] Non-limiting, the yeast extract described above may be any one of yeast extracts A, B, C, or D described in the later examples, or a mixture thereof. In one embodiment, the yeast extract may be yeast extract A or B described in the later examples, or a mixture thereof. In another embodiment, the yeast extract may be yeast extract A described in the later examples. Specific examples include yeast extracts YK-21NL, HIMAX PR, YP21-CM, and yeast extract 21-NYP (all sold by Fuji Foods Industry Co., Ltd.).

[0148] The content of the yeast extract in the above-mentioned basal culture medium composition for animal cell culture is not particularly limited. The yeast extract content is preferably a concentration that achieves the desired animal cell proliferation effect and a concentration that does not cause proliferation impairment (due to excessive concentration). In the above-mentioned basal culture medium composition for animal cell culture, when the basal culture medium composition is set to 100, the content of the yeast extract is non-limitingly 0.01 to 25.0 parts by weight, 0.05 to 20.0 parts by weight, 0.10 to 15.0 parts by weight, 0.20 to 10.0 parts by weight, 0.25 to 5.0 parts by weight, or 0.25 g to 2.0 parts by weight. In one embodiment, in the above-mentioned basal culture medium composition for animal cell culture, when the basal culture medium composition is set to 100, the content of the yeast extract is 0.05 to 20.0 parts by weight.

[0149] It can be used in combination with the yeast extract described above, using extracts from natural sources other than yeast extract as components of the composition for the basal culture medium of the above-mentioned animal cell culture. The above-mentioned extracts from natural sources may, without limitation, include extracts from, for example, cereals such as corn, wheat, barley, and rice; legumes such as kidney beans and peas (excluding soybeans); animal tissues such as meat and bone; or microbial tissues such as bacteria and mycelia.

[0150] In one approach, the extracts from natural sources other than the yeast extract mentioned above do not contain soybean hydrolysate.

[0151] The types of inorganic salts included in the above-mentioned composition for basal culture medium for animal cell culture are not particularly limited. Salts of calcium, iron, potassium, magnesium, sodium, zinc, etc., are included without limitation. Salts include chlorides, sulfates, phosphates, hydrochlorides, bicarbonates, nitrates, etc., without limitation. Inorganic salts include calcium chloride, ferric chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate, ferric sulfate, ferric nitrate, magnesium chloride, zinc sulfate, etc. In one embodiment, the above-mentioned inorganic salts include calcium chloride. In one embodiment, the above-mentioned composition for basal culture medium for animal cell culture may contain two or more inorganic salts.

[0152] In one embodiment, the above-mentioned composition for the basal culture medium of animal cell culture comprises two or more inorganic salts selected from calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate. In another embodiment, the above-mentioned composition for the basal culture medium of animal cell culture comprises calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate.

[0153] The amount of the inorganic salts contained in the above-mentioned basal culture medium composition for animal cell culture is not particularly limited. A suitable concentration for animal cell culture can be appropriately adopted depending on the type of inorganic salt.

[0154] Sugars are the initial oxidation products of polyols, possessing either a formyl group (-CHO) or a carbonyl group (>C=O). Sugars with a formyl group are classified as aldoses, while those with a carbonyl group are classified as ketoses. Sugars are abundant in plant tissues, honey, and fruits; glucose, in particular, is a vital energy source for organisms.

[0155] A substance formed by two monosaccharide molecules linked by a glycosidic bond is called a disaccharide; a substance formed by three monosaccharide molecules linked by a glycosidic bond is called a trisaccharide; and similarly, a substance formed by four monosaccharide molecules linked by a glycosidic bond is called a tetrasaccharide. A substance formed by the linkage of approximately 20 monosaccharide molecules is called an oligosaccharide. A substance formed by the polymerization of multiple monosaccharides through glycosidic bonds is called a polysaccharide. A substance in which the hydroxyl group of a sugar is replaced by a hydrogen atom is called a deoxysugar; a substance in which the terminal carbon of an aldose is replaced by a carboxyl group is called a uronic acid; a substance in which the hydroxyl group is replaced by an amino group is called an amino sugar; and a substance in which a ketone or aldehyde group is reduced to an alcohol is called a sugar alcohol.

[0156] Examples of sugars include monosaccharides such as glucose, mannose, fructose, galactose, and xylose. Examples of disaccharides composed of two glucose molecules include trehalose, isotrehalose, maltose, cellobiose, and isomaltose. Other known disaccharides include lactose (β-galactose and β-glucose are bonded by a 1,4-galactoside bond) and sucrose (α-glucose and β-fructofuranose (fructose) are bonded by a 1,2-glycosidic bond).

[0157] The types of sugars contained in the above-mentioned basal culture medium composition for animal cell culture are not particularly limited. Glucose, fructose, trehalose, galactose, sucrose, or maltose are included without limitation. In one embodiment, the sugar contained in the above-mentioned basal culture medium composition for animal cell culture is glucose. In one embodiment, the above-mentioned basal culture medium composition for animal cell culture may contain two or more sugars.

[0158] The amount of the sugars contained in the above-mentioned basal culture medium composition for animal cell culture is not particularly limited. A suitable concentration for animal cell culture can be appropriately adopted depending on the type of sugar.

[0159] In one embodiment, the composition for the basal culture medium of the animal cell culture described above may further contain vitamins.

[0160] "Vitamins" are a collective term for organic compounds other than carbohydrates, proteins, and lipids that an organism cannot synthesize in sufficient quantities to meet its micronutrient requirements for survival and growth. Vitamins are classified according to their function, not their name. For example, vitamin A includes retinaldehyde and retinol. Vitamins can be classified as fat-soluble vitamins and water-soluble vitamins. Below are examples of vitamins in humans.

[0161] fat-soluble vitamins

[0162] Vitamin A: Retinol, beta-carotene, alpha-carotene, beta-cryptoxanthin, etc.

[0163] Vitamin D: Ergocalciferol, Cholecalciferol

[0164] Vitamin E: Tocopherol, Tocotrienol

[0165] Vitamin K: Phloroquinone and Menaquinone, two naphthoquinone derivatives

[0166] Water-soluble vitamins

[0167] Vitamin B complex

[0168] Vitamin B1: Thiamine

[0169] Vitamin B2: Riboflavin, also known as Vitamin G.

[0170] Vitamin B3: Niacin. Also known as vitamin PP.

[0171] Vitamin B5: Pantothenic acid

[0172] Vitamin B6: Pyridoxal, Pyridoxamine, Pyridoxine

[0173] Vitamin B7: Biotin, also known as vitamin Bw or vitamin H.

[0174] Vitamin B9: Folic acid, also known as vitamin Bc or vitamin M.

[0175] Vitamin B 12 : Cyanocobalamin, Methylcobalamin, Hydroxycobalamin

[0176] Vitamin C: Ascorbic Acid

[0177] Sometimes, substances that do not meet the above definition of vitamins but have similar functions are called "vitamin-like substances." Vitamin-like substances include those historically mistakenly considered vitamins or those no longer considered vitamins due to changes in the definition. Examples of vitamin-like substances are listed below.

[0178] Vitamin B4: Adenine

[0179] Vitamin B8: Ergadenylic acid (Adenosine monophosphate)

[0180] Vitamin B 10 Vitamin R is a mixture of various B vitamins, with folic acid as the main component.

[0181] Vitamin B 11 Folic acid-like compounds.

[0182] Vitamin B 13 whey acid

[0183] Vitamin B 14 : A mixture of folic acid or lipoic acid, etc.

[0184] Vitamin B 15 Pan-amino acid

[0185] Vitamin B 16 Dimethylglycine

[0186] Vitamin B 17 amygdalin

[0187] Vitamin B H Inositol

[0188] Vitamin B P :choline

[0189] Vitamin B T Carnitine

[0190] Vitamin B X Para-aminobenzoic acid (part of the structure of folic acid, also known as PABA)

[0191] Vitamin F: essential fatty acids such as linoleic acid

[0192] Vitamin I: Rice bran extract.

[0193] Vitamin J: Catechol, flavonoids, or choline

[0194] Vitamin L1: anthranilic acid

[0195] Vitamin L2: Adenylthiomethylpentose

[0196] Vitamin N: Thioctic acid (α-lipoic acid)

[0197] Vitamin O: Carnitine

[0198] Vitamin P: Flavonoids such as quercetin, hesperidin, rutin, and senna

[0199] Vitamin Q: ubiquinone

[0200] Vitamin S: Salicylic acid

[0201] Vitamin T: Tegotin

[0202] Vitamin U: Methylmethionine sulfonium chloride (also known as "Cabagin").

[0203] Vitamin V: Nicotinamide adenine dinucleotide

[0204] The types of vitamins contained in the above-mentioned basal culture medium compositions for animal cell culture are not particularly limited. In this specification, unless otherwise specified, "vitamin" includes the aforementioned vitamin-like substances.

[0205] The vitamin contained in the above-mentioned composition for basal culture medium for animal cell culture is not limited to one or more vitamins selected from pantothenate (e.g., calcium pantothenate), folic acid, pyridoxine or a salt thereof (e.g., pyridoxine hydrochloride), riboflavin, thiamine or a salt thereof (e.g., thiamine hydrochloride), choline (e.g., glycerophosphatidylcholine), inositol (e.g., myo-inositol), and nicotinamide (vitamin V). In one embodiment, the above-mentioned composition for basal culture medium for animal cell culture contains all of the following substances: pantothenate (e.g., calcium pantothenate), folic acid, pyridoxine or a salt thereof (e.g., pyridoxine hydrochloride), riboflavin, thiamine or a salt thereof (e.g., thiamine hydrochloride), choline (e.g., glycerophosphatidylcholine), inositol (e.g., myo-inositol), and nicotinamide (vitamin V).

[0206] The amount of the aforementioned vitamins contained in the above-mentioned basal culture medium composition for animal cell culture is not particularly limited. A suitable concentration for animal cell culture can be appropriately adopted depending on the type of vitamin.

[0207] In addition to yeast extract, amino acids, inorganic salts, vitamins, and sugars, the above-mentioned composition for basal culture medium for animal cell culture may also contain other components. These other components can be any ingredients that can be used in the composition for basal culture medium for animal cell culture, such as pH indicators (phenol red), glutathione, fatty acids such as linoleic acid, and thymidine.

[0208] "Basic culture medium for animal cells" refers to a culture medium that serves as the basis for culturing, proliferating, and / or differentiating animal cells, and is generally used with the appropriate addition of serum, etc., without limitation. In this specification, unless otherwise specified, "culture" is used to include the meaning of proliferation. In this specification, "composition for basic culture medium for animal cell culture" refers to a composition that serves as the basis for a basic culture medium for culturing animal cells. The composition for basic culture medium for animal cell culture can be a liquid composition or a solid composition. For example, a liquid composition for animal cell culture can be used directly as a basic culture medium for animal cells. Alternatively, it can be a paste after water removal, preserved and sold by setting appropriate water activity, and then used to prepare a basic culture medium for animal cells by adding an appropriate amount of water. Alternatively, it can be a solid composition, preserved and sold as a powder raw material that has undergone drying treatment, etc., and dissolved in a solvent (e.g., water) to prepare a basic culture medium for animal cells before use. In this specification, "basic culture medium for animal cells" or "basic culture medium" is sometimes used with the same meaning as "composition for basic culture medium for animal cell culture."

[0209] There is no particular limitation on the species of animal from which the animal cells are derived. In one embodiment, the animal cells are cells from vertebrates such as mammals. Vertebrates can be any animal among mammals, reptiles, birds, and amphibians, including, for example, humans, cattle, pigs, rabbits, chickens, sheep, and goats. In another embodiment, the vertebrate is a mammal other than a human. In another embodiment, the animal cells are cells derived from oviparous animals that lay eggs with eggshells, such as birds or reptiles. In yet another embodiment, the animal cells are chicken cells.

[0210] There is no particular limitation on the type of animal cell used. For example, it can be any of the following: somatic cells of adult origin, embryos, fertilized eggs, cultured cells with established cell lines, etc. Without limitation, for example, in the case of chickens, embryos can be used, and embryos of 0-21 days, 2-19 days, 4-17 days, and 6-15 days are preferred.

[0211] There are no particular limitations on the tissues derived from animal cells. The aforementioned animal cells are not limited to cells derived from muscle, skin, intestine, heart, brain, stomach, embryonic membrane, liver, kidney, or lung.

[0212] In the following formulation, the amino acids are cystine and / or cysteine, and glutamine, with cystine and / or cysteine, and glutamine as the main components. Alternatively, amino acids other than cystine and / or cysteine, and glutamine may not be included, but this is not a limitation. Cystine and / or cysteine, and glutamine are essential elements of the above-mentioned composition for the basal culture medium of animal cells, and it is conceivable that combining other amino acids may nutritionally promote their growth.

[0213] In one embodiment, the present invention relates to the use of yeast extract in a composition of a basal culture medium for animal cell culture, which is composed of yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids are cystine and / or cysteine, and glutamine.

[0214] In one embodiment, the present invention relates to a combination of yeast extract, amino acids, inorganic salts and sugars for use in a composition for a basal culture medium for animal cell culture, wherein the amino acids are cystine and / or cysteine, and glutamine.

[0215] In one embodiment, the present invention relates to a kit for manufacturing a composition for a basal culture medium for animal cell culture, comprising yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids are cystine and / or cysteine, and glutamine. In the above kit, the yeast extract, amino acids, inorganic salts, and sugars may exist individually or in a mixture of two or more components prior to manufacturing the composition for the basal culture medium for animal cell culture.

[0216] 2. Animal cell culture methods

[0217] In one embodiment, the present invention relates to a method for culturing animal cells.

[0218] The above-mentioned culture method includes, without limitation, culturing animal cells in a basal culture medium for animal cells, wherein the basal culture medium for animal cells contains yeast extract, amino acids, inorganic salts and sugars, and the amino acids include L-cysteine ​​and / or L-cysteine, and L-glutamine.

[0219] The culture conditions described above for animal cells are not particularly limited. Appropriate culture conditions can be adopted depending on the species and tissue of the animal from which the animal cells are derived.

[0220] In one embodiment, the yeast extract is an extract of yeast with a high amino acid profile.

[0221] In one embodiment, the yeast extract is an extract of a low-nucleic acid type of yeast.

[0222] In one embodiment, the yeast extract is an extract of yeast with a high amino acid profile and low nucleic acid profile.

[0223] In one embodiment, the basal culture medium for animal cells used in the above-described animal cell culture method may contain vitamins.

[0224] The meanings of “yeast extract,” “amino acid,” “inorganic salt,” “vitamin,” “sugar,” and “basal culture medium for animal cell culture” are as described in “1. Composition of basal culture medium for animal cell culture.”

[0225] The information described in “1. Composition for basic culture medium for animal cell culture” can also be applied to the culture method described in this document, provided there are no particular contradictions.

[0226] In one embodiment, the present invention relates to a culture medium for a method of culturing animal cells, comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids comprise L-cysteine ​​and / or L-cysteine, and L-glutamine.

[0227] In one embodiment, the present invention relates to the use of a culture medium in a method of culturing animal cells, the culture medium comprising yeast extract, amino acids, inorganic salts and sugars, the amino acids comprising L-cysteine ​​and / or L-cysteine, and L-glutamine.

[0228] 3. Animal cells and processed products

[0229] In one embodiment, the present invention comprises animal cells obtained by culturing them using the animal cell culture method of the present invention.

[0230] In one embodiment, the present invention relates to processed food containing the aforementioned animal cells.

[0231] The meanings of "animal cell culture method" and "animal cell" are as described in "1. Composition of basic culture medium for animal cell culture" and "2. Animal cell culture method".

[0232] The information described in “1. Composition for basic culture medium for animal cell culture” and “2. Method for culturing animal cells” can also be applied to the animal cells and processed foods described in this item, provided there are no particular contradictions.

[0233] Animal cells and processed foods obtained by culturing using the animal cell culture method of the present invention can be produced with a lower environmental burden compared to the use of conventional commercially available basal culture medium compositions. Therefore, as a result, low-cost animal cells and processed foods with a low environmental impact can be obtained. Furthermore, they possess the following characteristics: a more complex flavor that cannot be obtained by animal cell culture methods, and the unique meat flavor inherent in the original animal tissue.

[0234] "Processed foods" include, for example, foods containing cultured animal cells, or meat formed from cultured animal cells (cultured meat). Cultured meat can be used as an ingredient in the same way as ordinary meat, for example, in the form of beef, pork, rabbit, chicken, sheep, goat, etc. The term "food" in "processed foods" includes either raw ingredients before cooking or cooked food (meals). The term "processing" in "processed foods" refers not only to simply cooking ingredients to prepare meals, but also to all processing of ingredients as described below: pre-processing ingredients to create more processed ingredients; or processing ingredients to provide new ingredients with adjusted flavors, properties, etc.

[0235] Example

[0236] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments. Those skilled in the art can readily make modifications / changes to the present invention based on the description herein, and these modifications / changes are included within the technical scope of the present invention.

[0237] Example 1 Study on the minimum required added amino acid species

[0238] In this embodiment, the following were investigated: when culturing animal cells, if yeast extract is used as an amino acid source, can the cells proliferate using only yeast extract without adding additional amino acids, and what amino acids are required at the minimum level if they cannot proliferate.

[0239] (method)

[0240] AA-IMEM buffer was prepared by dissolving the components of Duchenne Modified Eagle Medium (DMEM) in ion-exchanged water after removing the amino acids. (Table 1)

[0241] Table 1 Composition of AA-IMEM buffer

[0242]

[0243] The following seven yeast extracts (B, D, E, F, H, I, and J) were further added to the AA-IMEM buffer to bring the final concentration in the culture medium to 1 g / L.

[0244] Table 2 Yeast extract added in Example 1

[0245]

[0246] Cell culture experiments were conducted by adding various amino acids considered necessary for cell proliferation in DMEM medium to “AA-IMEM buffer + yeast extract” according to conditions 1-16 in Table 3. This investigated the essential amino acids required for cell proliferation when using yeast extract as the culture material. The amount of each amino acid added was equal to the amount contained in the DMEM medium. Additionally, a medium with the same amino acid composition as DMEM medium was used as PC (positive control), a medium with the same amino acid composition as DMEM medium but with 1 / 8 of the added amount was used as condition 19, and a medium with 1 / 32 of the added amount was used as condition 20. All reagents used in the experiments were food additive grade.

[0247] Table 3 Amino acid conditions of the experimental culture medium

[0248]

[0249] Cell culture experiments were conducted by adding 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphoteric acid (PSA) to AA-IMEM buffer + yeast extract and amino acids under the conditions in Table 3.

[0250] The mouse-derived myoblast cell line C2C12 was used in the cell culture assay. C2C12 cells were cultured to a density of 5 × 10⁶ cells / year. 4 Cells were seeded in 12-well plates using a per-well method and cultured for 3 days in a CO2 incubator with various culture media at 37°C. Cell adhesion and proliferation were observed after culture under each condition. Furthermore, images of the cultured cells were taken to confirm their growth status.

[0251] (result)

[0252] The results of the cell images are shown in Figure 1 .according to Figure 1 Cell images were used, and cases where the proportion of cells on the bottom surface of the culture vessel was more than 50% were marked as ○, cases where the proportion of cells was 10-50% were marked as △, and cases where no cell adhesion was observed were marked as ×. The results are shown in Table 4.

[0253] Table 4. Cell proliferation scores under various conditions

[0254]

[0255] like Figure 1 As shown in Table 4, cell adhesion and subsequent proliferation were observed when all amino acids were added (PC) and when 1 / 8 of the total amount was added (condition 19). In contrast, cell adhesion was not observed when 1 / 32 of all amino acids were added (condition 20) and when no amino acids were added (condition 1). This clearly demonstrates that when using only yeast extract (final concentration in the culture medium of 1 g / L), the amino acids required for cell adhesion and proliferation are insufficient, and further amino acid supplementation is necessary.

[0256] The required amino acids in DMEM medium were added individually to investigate the need for additional amino acids besides yeast extract. The results showed that for most amino acids, cell adhesion was not observed even when added individually. In contrast, cell adhesion was observed only under condition 15 with the addition of L-cysteine, confirming that the addition of L-cysteine ​​to yeast extract can improve cell adhesion.

[0257] Furthermore, with the addition of more than 1 / 8 of all amino acids (condition 19, PC), cells proliferated and attached. In contrast, under condition 18, where only L-glutamine was removed, the proportion of attached cells was significantly reduced, thus confirming that L-glutamine is essential for cell proliferation. On the other hand, under condition 16, where only L-glutamine was added, no cell attachment was observed, indicating that L-glutamine alone is insufficient, and L-cysteine ​​is also required. Based on the above results, it is clear that the minimum required amino acids in the basal culture medium are L-cysteine ​​and L-glutamine.

[0258] Furthermore, cell adhesion and proliferation were confirmed under condition 19, while cell adhesion was virtually nonexistent under condition 20, indicating that at least 6.13 mg / L of L-cysteine ​​and 74.5 mg / L of L-glutamine are required.

[0259] Example 2 Culture test of multiple cell species

[0260] Example 1 showed that L-cysteine ​​and L-glutamine were required for the culture of C2C12 cells. In this example, the necessity of these amino acids, not only in the mouse-derived myoblast cell line C2C12 but also in other cell lines, was verified. Cell culture experiments were performed using MDBK cells (bovine kidney-derived cell line) and HDFa cells (adult skin fibroblast cell line) in the same manner as in Example 1.

[0261] (method)

[0262] The amino acid-free components of the DMEM medium were dissolved in deionized water to prepare AA-IMEM buffer (Table 1). Equal volumes of the yeast extracts used in Example 1 were mixed to prepare a yeast extract mix. This yeast extract mix was then added to the medium to achieve a final concentration of 1 g / L. L-cysteine ​​or L-cysteine ​​and L-glutamine were added as shown in Table 5 to confirm the cell growth status of each cell. It should be noted that in the cell culture experiments, 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphoteric acid (PSA) were further added to the AA-IMEM buffer + yeast extract and the amino acids listed in Table 5.

[0263] Table 5 Amino acid conditions of the experimental culture medium

[0264]

[0265] The number of MDBK cells and HDFa cells was increased to 5 × 10⁸. 4 Cells were seeded in 12-well plates using a per-well method and cultured in a CO2 incubator at 37°C. For MDBK cells, the state of each cell was photographed on day 5 of culture, and for HDFa cells, the state of each cell was photographed on day 4 of culture to confirm cell adhesion.

[0266] (result)

[0267] The results of the cell images are shown below. Figure 2 .according to Figure 2 Cell images were used, and cases where the proportion of cells on the bottom surface of the culture vessel was more than 50% were marked as ○, cases where the proportion of cells was 10-50% were marked as △, and cases where no cell adhesion was observed were marked as ×. The results are shown in Table 6.

[0268] Table 6. Cell proliferation scores under various conditions

[0269]

[0270] like Figure 2 As shown in Table 6, no cell adhesion was observed under condition 1, where no amino acids were added to any cells but only yeast extract mix was added. In contrast, cell adhesion was observed under condition 2, where L-cysteine ​​was added. Furthermore, cell proliferation was confirmed under condition 3, where both L-cysteine ​​and L-glutamine were added.

[0271] These results confirm that L-cysteine ​​and L-glutamine significantly contribute to cell adhesion and proliferation. It is also demonstrated that by including L-cysteine, L-glutamine, and yeast extract in the culture medium, cell culture can be performed in at least the three cell lines investigated in Examples 1 and 2.

[0272] Example 3 Study on added yeast extract Confirmation test of cell proliferation adaptability of various yeast extracts Example 4 Study on added yeast extract Selection of optimal yeast extract

[0273] In this embodiment, in order to select the most suitable yeast extract to be added to the culture medium, a cell proliferation adaptability confirmation test was conducted using a variety of yeast extracts with different characteristics.

[0274] (method)

[0275] The basic culture medium with the composition shown in Table 7 was prepared.

[0276] Table 7. Basal culture medium used in the experiment

[0277]

[0278] Eight yeast extracts (A-H) from Table 8 were added to the culture medium to achieve final concentrations of 0.25 g / L, 0.5 g / L, 1.0 g / L, and 2.0 g / L, respectively. All eight yeast extracts were derived from the yeast genus and exhibited the characteristics shown in Table 8.

[0279] Table 8. Types of yeast extracts used

[0280]

[0281] Duck liver-derived cells were used as primary cultured cells in the experiment. After being harvested from the organism, the duck liver-derived cells were cultured up to the third passage in a basal medium with the same composition as DMEM medium, supplemented with 10% serum and 1% PSA. The duck liver-derived cells were cultured at a concentration of 1.5 × 10⁻⁶ cells / year. 4 Cells were seeded in 96-well plates using a cell / well ratio, and cell culture assays were performed with 10% serum and 1% PSA added to each culture medium.

[0282] In cell proliferation, Incucyte SX5 (Sartorius) was used to record the proportion of cells in the observed images as the cell occupancy percentage over time in each culture medium. The recorded data was fitted with logistic curves to calculate the midpoint of cell proliferation (Exponental Growth 50%: EG50), and cell proliferation rates were compared. Yeast extracts that reached EG50 after more than 100 hours were considered to have cell proliferation inhibitory capabilities.

[0283] In addition, DMEM medium (FUJIFILM) was used in PC, and a medium without yeast extract was used in NC, which is the basal medium described in Table 6.

[0284] (result)

[0285] The EG50 arrival times of cells cultured in various media are shown in Table 9. A comparison of the EG50 values ​​in Table 9 reveals that yeast extracts B, E, and F, when added at high concentrations (2.0 g / L and 1.0 g / L), exhibited decreased cell proliferation. These proliferation inhibitions were reduced by setting the concentrations to low. Furthermore, in yeast extracts F, H, and G (which did not exhibit cell proliferation inhibition at high concentrations), even at concentrations below 0.5 g / L, the EG50 arrival time was over 100 hours, indicating a decrease in cell proliferation rate. These results confirm that these yeast extracts inhibit cell proliferation even at low concentrations.

[0286] Table 9. EG50 arrival time (h) of cells cultured in various culture media.

[0287]

[0288] Figure 3

[0289] In this embodiment, an experiment was conducted to screen for the yeast extract most suitable for cell proliferation. The yeast extracts used in the screening were yeast extracts A, B, C, and D, excluding the yeast extract that was confirmed to have cell proliferation inhibitory ability at low concentrations in Example 3.

[0290] (method)

[0291] Duck liver-derived cells were used as primary cultured cells in the experiment. After being harvested from the organism, the duck liver-derived cells were cultured in a basal medium with the same composition as DMEM medium, supplemented with 10% serum and 1% PSA, until passage 5-6. Repeated passages were performed to investigate cell proliferation.

[0292] In the experimental basal culture medium listed in Table 7, yeast extracts were added to achieve a final concentration of 1.0 g / L, and 10% serum and 1% PSA were further added to prepare a complete culture medium.

[0293] The duck liver-derived cells were used to achieve a density of 1.5 × 10⁻⁶. 5Cells were seeded in 60 mm culture dishes using the cell / dish method and cultured. Additionally, during passage, a portion of the cells were cultured at a density of 5 × 10⁶ cells / mL. 3 Cells were seeded in 96-well plates using a cell / well ratio, and the number of viable cells was compared by measuring absorbance at 450 nm using a Cell Counting Kit-8 (Dongjin Chemical Research Institute). Cultures were performed up to the third generation, and the number of viable cells when cultured with each yeast extract was compared by measuring absorbance.

[0294] (result)

[0295] In the NC (negative control) without yeast extract, cells did not proliferate, therefore second-generation culture was not possible. On the other hand, it was confirmed that in the medium supplemented with yeast extract, cell proliferation occurred through passage culture of duck liver-derived cells. Furthermore, it was confirmed that cell proliferation was significantly greater in media supplemented with yeast extracts A, B, and C compared to media supplemented with other yeast extracts. Example 5 Optimization of yeast extract-added culture medium )

[0296] Figure 4

[0297] In this embodiment, yeast extracts A and B, which showed particularly good cell proliferation in Example 5, were used to investigate the optimal composition of the culture medium.

[0298] (method)

[0299] Duck liver-derived cells were used as primary cultured cells in the experiment. After being harvested from the organism, the duck liver-derived cells were cultured in a basal medium with the same composition as DMEM medium, supplemented with 10% serum and 1% PSA, until passage 5-6. Repeated passages were performed to investigate cell proliferation.

[0300] Complete culture media were prepared by adding 2.0 g / L of each yeast extract, 10% serum, and 1% PSA to the basal culture medium listed in Table 10. In this experiment, calcium chloride was further added to the basal culture medium composition in Table 7 of Example 4 in the same amount as that contained in DMEM culture medium (Table 10).

[0301] Table 10. Basal culture media used in the experiment

[0302]

[0303] The duck liver-derived cells were used to achieve a density of 1.5 × 10⁻⁶. 5 Cells were seeded in 60 mm culture dishes using the cell / dish method and cultured. Additionally, during passage, a portion of the cells were cultured at a density of 5 × 10⁶ cells / mL.3 Cells were seeded in 96-well plates using a cell / well ratio, and the number of viable cells was compared by absorbance measurement at 450 nm using a Cell Counting Kit-8 (Dongjin Chemical Research Institute). Cultures were performed up to the 5th generation, and the number of viable cells when cultured with each yeast extract was compared by absorbance value.

[0304] It should be noted that the PC (positive control) used a medium with the same composition as the DMEM medium, while the NC (negative control) was set to a medium containing only the experimental basal medium with the composition shown in Table 10 and without the addition of yeast extract.

[0305] (result)

[0306] The results are shown in Example 6 Confirmation test 1 of the universality of the yeast extract-added culture medium It was determined that increasing the amount of yeast extract to 2.0 g / L and adding calcium chloride to the basal medium improved culturability. It was confirmed that NC cells ceased proliferation at passage 3 and could not be cultured thereafter, but in media with added yeast extract, PC cells maintained approximately 70% or more of their culturability at passage 5.

[0307] Figure 5

[0308] In this embodiment, yeast extract A, which showed particularly good cell proliferation in Example 5, and yeast extract AB, which is a mixture of equal amounts of A and B, were used to conduct culture experiments on cells derived from various organs of chickens, and to confirm the universality of these yeast extracts.

[0309] (method)

[0310] Cells derived from chicken liver, muscle, lung, kidney, and brain were used as primary cultured cells in the experiment. Cells from various chicken organs were cultured in their respective culture media after being removed from the organism. It should be noted that the yeast extract-added culture medium used the following composition: The experimental basal medium with the composition shown in Table 10 of Example 5 was prepared by adding the vitamins contained in DMEM medium (calcium pantothenate, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, glycerophosphate choline, myo-inositol, and nicotinamide as shown in Table 1) to achieve the same composition as DMEM medium, and adding 2.0 g / L of yeast extract A. Additionally, a basal medium with the same composition as DMEM medium was used as a positive control. These media were used in the experiment after adding 10% serum and 1% PSA.

[0311] Cells derived from various chicken organs were inoculated into yeast extract-supplemented medium and positive control medium, respectively. Subcultures were performed every 3-4 days, and cell counts were recorded at the third passage time point. The optimal cell count was defined as 3 × 10⁶ cells / year at passage. 5 Cells were seeded in a 6-well plate using a cell / well method.

[0312] (result)

[0313] The results are shown in Example 7 Confirmation test 2 of the universality of the yeast extract-added culture medium For cells cultured in yeast extract A-added medium from various chicken organ sources, growth was confirmed to be on par with the positive control. Therefore, it is clear that yeast extract-added medium maintains good cultureability for cells from various organ sources. Furthermore, in yeast extract AB-added medium, slightly reduced cultureability was observed in the lung / kidney / brain cases, while in the liver / muscle cases, cultureability was observed to be on par with PC and yeast extract A-added medium.

[0314] Figures 6 to 8

[0315] In this embodiment, yeast extract A, which exhibits excellent proliferation properties as described in Example 6, was used to conduct culture experiments on cell lines and primary cultured cells from different sources, confirming its culturability.

[0316] The experiments used cell lines RL34 (rat normal liver-derived cells), MDBK (bovine normal kidney-derived cells), and primary cultured bovine muscle-derived cells. The yeast extract-added culture medium used the following composition, with DMEM medium as a positive control: The basal medium of the composition shown in Table 10 of Example 5 was prepared by adding the vitamins contained in DMEM medium (calcium pantothenate, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, glycerophosphate choline, myo-inositol, and nicotinamide from Table 1) to achieve the same composition as DMEM medium, and adding 2.0 g / L of yeast extract A. These media were then used in the experiments after adding 10% inactivated FBS and 1% PSA.

[0317] (method)

[0318] Cells cultured in DMEM medium were divided into RL34 cells at 3 × 10⁶ cells per cell line. 5 Cells, MDBK cells 2×10 5 Cells, bovine muscle-derived cells 3×10 5 Cells were seeded in 6-well plates and cultured in various media. Cells were passaged every 3–4 days and counted.

[0319] (result)

[0320] The results are shown in Example 8 Study on the lower limit range of the concentration of added amino acids in the base culture medium For cells cultured in any cell type with yeast extract A added to the medium, it was confirmed that more than 70% of the cultureability was maintained compared to DMEM medium used as a positive control.

[0321] Figure 9

[0322] In this embodiment, the concentration range of amino acids (L-glutamine, L-cysteine) added to the basal culture medium, especially the minimum required concentration, was studied.

[0323] (method)

[0324] MDBK cells were cultured in DMEM medium to reach 1×10⁻⁶ cells / year. 5 Cells were seeded in 12-well plates at a ratio of 2 ml of culture medium per well and cultured. Subcultures were performed every 3–4 days, and the viable cell count was measured at the end of the third subculture.

[0325] It should be noted that each culture medium was used for the experiment with 10% inactivated FBS and 1% PSA added.

[0326] Cells used: MDBK cells (cell line)

[0327] Cell count before culture: 1×10 5 cell

[0328] Amino acid concentration range: 0.002~0.063% by mass

[0329] Set 0.063% by mass as 1, and halve the amount of amino acids added each time from this amount onwards, setting the minimum amount of amino acids added as 0.002% by mass. The composition of the basal culture medium is shown in the table below.

[0330]

[0331] Cell proliferation results after culturing in the above-mentioned basal medium are shown in... Figure 9 . Figure 9 The horizontal axis represents the mass fraction (w / v, %) of the amino acids (L-glutamine, L-cysteine) contained in the basal medium relative to the mass fraction (w / v, %) when the basal medium is set to 100. Example 9 Confirmation of culture when L-cysteine is replaced with L-cysteine The vertical axis represents the number of viable cells at the end of the third passage culture.

[0332] (result)

[0333] At the second generation time point, even with the amino acid addition reduced to 0.002% by mass, cell proliferation in MDBK cells was confirmed. Furthermore, at the third generation time point, although cell proliferation was not observed at 0.002% by mass, this was a sustainable result. Additionally, cell proliferation was confirmed at 0.004% by mass or higher at the third generation.

[0334] Based on the above results, when the basal culture medium is set to 100, the lower limit of the concentration range of L-cysteine ​​and L-glutamine is 0.002 by mass.

[0335] ​

[0336] In this embodiment, the culturing performance was confirmed when L-cysteine ​​was replaced with L-cysteine.

[0337] In this embodiment, the culture properties of L-cysteine ​​hydrochloride monohydrate, which has higher solubility than L-cysteine, were studied from the perspective of comparison with L-cysteine ​​when L-cysteine ​​hydrochloride monohydrate was used as an amino acid added to the basal medium.

[0338] (method)

[0339] Cells used: MDBK cells (cell line), RL34 cells (cell line), duck liver-derived cells (primary culture cells)

[0340] Cell count before culture: MDBK and RL34 cells = 1 × 10⁻⁶ 5 Cells / well, duck liver-derived cells = 1 × 10⁻⁶ 6 Cells / 10cm culture dish

[0341] L-cysteine ​​hydrochloride monohydrate addition amount: Since L-cysteine ​​is a dimer of L-cysteine, it was added in a manner that was twice the molar amount of L-cysteine.

[0342] The composition of the basal culture medium is shown in the table below.

[0343]

[0344] MDBK and RL34 cells were passaged in a complete medium supplemented with 1% PSA and 10% FBS. Additionally, duck liver-derived cells were passaged in a complete medium supplemented with 1% PSA and 10% serum. Passage was performed every 3-4 days until the third pass.

[0345] (result)

[0346] The results at the end of the third passage culture are shown in Figure 10. The vertical axis of Figure 10 represents the number of viable cells. Culture experiments were conducted using MDBK cells (cell lines) and RL34 cells as cell lines, and duck liver-derived cells as primary culture cells. The results showed that, under any circumstances, there was essentially no difference in cultureability due to the replacement of L-cysteine ​​with L-cysteine ​​hydrochloride monohydrate.

[0347] This result clarifies that using either L-cysteine ​​or L-cysteine ​​as an added amino acid will not affect cell culture.

[0348] It should be noted that the same results were obtained when L-cysteine ​​was added twice as much (0.14%).

[0349] Industrial applicability

[0350] This invention provides a basic culture medium for animal cells that reduces costs and environmental impact. By utilizing the culture medium and animal cell culture method of this invention, processed foods containing animal cells can be provided in a cheaper and less environmentally burdensome manner.

Claims

1. A composition for a basal culture medium for animal cell culture, comprising yeast extract, amino acids, inorganic salts, and sugars, wherein, The amino acids in the composition include cystine and / or cysteine, as well as glutamine.

2. The composition for basal culture medium for animal cell culture according to claim 1, wherein, When the composition for the basal culture medium is set to 100, the total amino acid content in the composition is 0.002~1.0 parts by mass.

3. The composition for basal culture medium for animal cell culture according to claim 1 or 2, wherein, The proportions of cystine and / or cysteine, and glutamine, relative to all amino acids in the composition, are 50% or more.

4. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 3, wherein, When the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine ​​and glutamine in the composition is 0.001~1.0 parts by weight.

5. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 4, wherein, Based on the amount of solid components per unit of the composition for basal culture medium, the content of cystine and / or cysteine, and glutamine in the composition is 0.1 to 5.0 parts by weight.

6. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 5, wherein, When the composition for the basal culture medium is set to 100, the content of cystine and / or cysteine ​​in the composition is 0.0001~0.02 parts by mass.

7. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 6, wherein, Based on the amount of solid components per unit of the composition for basal culture medium, the content of cystine and / or cysteine ​​in the composition is 0.005 to 1.0 parts by weight.

8. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 7, wherein, When the composition for the basal culture medium is set to 100, the content of glutamine in the composition is 0.001~1.0 parts by mass.

9. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 8, wherein, The content of glutamine in the composition is 0.1 to 5.0 parts by weight per unit of solid component in the basal culture medium composition.

10. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 9, wherein, The yeast extract is a high-amino acid type yeast extract.

11. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 10, wherein, When the composition for the basal culture medium is set to 100, the total amino acid content of the yeast extract is 0.75 parts by mass or more.

12. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 11, wherein, The yeast extract is a low-nucleic acid yeast extract.

13. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 12, wherein, When the composition for the basal culture medium is set to 100, the total nucleic acid content of the yeast extract is less than 2.0 parts by mass.

14. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 13, wherein, The yeast extract is a high-amino acid type and low-nucleic acid type yeast extract.

15. The composition for basal culture medium for animal cell culture according to any one of claims 1 to 14, wherein, When the composition for the basal culture medium is set to 100, the content of the yeast extract is 0.05 to 20.0 parts by mass.

16. The composition for basal culture medium for animal cell culture according to claim 1 or 2, further comprising vitamins.

17. A method for culturing animal cells, the method comprising: Animal cells are cultured in a basal culture medium for animal cell culture, the basal culture medium for animal cell culture containing yeast extract, amino acids, inorganic salts and sugars, the amino acids being cystine and / or cysteine, and glutamine.

18. The cultivation method according to claim 17, wherein, The yeast extract is an extract of yeast with high amino acid content, yeast with low nucleic acid content, or yeast with both high amino acid content and low nucleic acid content.

19. An animal cell obtained by culturing using the culture method described in claim 17 or 18.

20. A processed food product comprising the animal cells of claim 19.

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