Composition for basal medium for animal cell culture

Abstract

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

Description

Composition for basal medium for animal cell culture

[0001] The present invention relates to a composition for a basal medium for animal cell culture and its use, a method for culturing animal cells, etc.

[0002] Cell culture is the growth and maintenance of cells isolated from living tissue in a culture medium. In recent years, research into the in vitro culture of animal cells, particularly animal cells, has progressed, including the development of cultured meat. The media used to culture animal cells generally consist of inorganic salts, carbohydrates, amino acids, vitamins, proteins, peptides, fatty acids, lipids, serum, etc.

[0003] "Cellular agriculture" is a technology that involves directly feeding nutrients to cells extracted from animals or plants to grow them and then producing them as food. Meat, fish, and other foods produced using cellular agriculture technology are collectively called "cellular foods." Cellular foods that fall under the category of "meat" are sometimes called "cultured meat."

[0004] Cell-based foods are being developed worldwide with a view to ensuring food security and reducing environmental impact. Two key factors for commercializing cell-based foods are that the ingredients and manufacturing process are suitable for food production, and that manufacturing costs are low. Integriculture Co., Ltd. (Bunkyo-ku, Tokyo, Japan) has successfully developed I-MEM 1.0, a basal medium composition—the most important element in the production of cell-based foods—using only approved food additive ingredients (Press Release PRTIMES: "Integriculture begins accepting orders worldwide for I-MEM, a proprietary basal medium composition made entirely from food ingredients, from Wednesday, October 26th"; https: / / prtimes.jp / main / html / rd / p / 000000025.000034252.html).

[0005] I-MEM 1.0 reduces production costs and makes the components of the culture medium food-grade, which has been a major challenge in the production of cell-cultured meat, etc. However, even I-MEM 1.0 is not sufficient in terms of reducing production costs, and there is a need for the development of cheaper and safer basal medium compositions.

[0006] JP 2005-532057 A describes an animal protein-free medium for culturing cells. Claims 1 and 2 of JP 2005-532057 A are as follows: [Claim 1] An animal protein-free cell culture medium comprising a soybean hydrolysate and a yeast hydrolysate. [Claim 2] The animal protein-free cell culture medium 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).

[0007] As can be understood from the descriptions of claims 1 and 2, the medium of JP-T-2005-532057 contains soybean hydrolysate as an essential ingredient. Furthermore, the main purpose of the cell culture in JP-T-2005-532057 is to produce biological products such as viruses and recombinant proteins in the cells (e.g., claim 23, paragraphs

[0011] and

[0012] of JP-T-2005-532057).

[0008] There has been a desire to develop a culture medium for animal cell culture that can be used as food, maintains cultivability, reduces the use of food additives, and has a low environmental impact.

[0009] Special Publication 2005-532057

[0010] PRTIMES Press Release: "Integriculture begins accepting orders worldwide on Wednesday, October 26th for its proprietary basal culture medium, I-MEM, made entirely from food ingredients"; https: / / prtimes.jp / main / html / rd / p / 000000025.000034252.html

[0011] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that by replacing part of the composition of Dulbecco's Modified Eagle's Medium (DMEM medium) with yeast extract and further adding cystine and / or cysteine, and glutamine, it is possible to maintain the same culturing properties as conventional DMEM medium while reducing the addition of amino acids, which are food additives, and thus arrived at the present invention.

[0012] The present invention includes, but is not limited to, the following aspects.

[0013] I [1] A composition for a basal medium for animal cells comprising yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids in the composition comprise cystine and / or cysteine, and glutamine. [2] The composition for a basal medium for animal cells according to [1], wherein the total amino acid content in the composition is 0.002 to 1.0 parts by mass, based on 100 parts of the basal medium composition. [3] The composition for a basal medium for animal cells according to [1] or [2], wherein the ratio of cystine and / or cysteine, and glutamine to the total amino acids in the composition is 50% or more. [4] The composition for a basal medium for animal cells according to any one of [1] to [3], wherein the content of cystine and / or cysteine, and glutamine in the composition is 0.001 to 1.0 parts by mass, based on 100 parts of the basal medium composition. [5] The composition for a basal medium for animal cells 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 mass per solid content of the basal medium composition. [6] The composition for a basal medium for animal cells according to any one of [1] to [5], wherein the content of cystine and / or cysteine ​​in the composition is 0.0001 to 0.02 parts by mass per 100 parts of the basal medium composition. [7] The composition for a basal medium for animal cells 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 part by mass per solid content of the basal medium composition. [8] The composition for a basal medium for animal cells according to any one of [1] to [7], wherein the content of glutamine in the composition is 0.001 to 1.0 part by mass per 100 parts of the basal medium composition. [9] The composition for a basal medium for animal cells according to any one of [1] to [8], wherein the glutamine content in the composition is 0.1 to 5.0 parts by mass per solid content of the basal medium composition.

[10] The composition for a basal medium for animal cells according to any one of [1] to [9], wherein the yeast extract is a high amino acid yeast extract.

[11] The basal medium composition for animal cells according to any one of [1] to

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

[12] The basal medium composition for animal cells according to any one of [1] to

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

[13] The basal medium composition for animal cells according to any one of [1 to

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

[14] The basal medium composition for animal cells according to any one of [1] to

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

[15] The basal medium composition for animal cells according to any one of [1] to

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

[16] A composition for a basal medium for animal cells according to [1] or [2], further comprising vitamins.

[17] A method for culturing animal cells, comprising culturing animal cells in a basal 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] The culture method according to

[17] , wherein the yeast extract is an extract of a high amino acid type yeast, an extract of a low nucleic acid type yeast, or an extract of a high amino acid type and low nucleic acid type yeast.

[19] Animal cells cultured by the culture method according to

[17] or

[18] .

[20] A processed food comprising the animal cells of

[19] .

[0014] II [1] A basal medium for animal cells comprising yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids are cystine and glutamine. [2] The basal medium for animal cells according to [1], wherein the amino acid content is 0.005 to 1.0 part by mass relative to 100 parts by mass of the basal medium. [3] The basal medium for animal cells according to [1] or [2], wherein the proportion of cystine and glutamine among the amino acids is 50% or more relative to 100 parts by mass of the basal medium. [4] The basal medium for animal cells according to [1] or [2], wherein the content of cystine and glutamine among the amino acids is 0.0025 to 1.0 part by mass relative to 100 parts by mass of the basal medium. [5] The basal medium for animal cells according to [1] or [2], wherein the yeast extract is a high-amino acid yeast extract. [6] The basal medium for animal cells according to [1] or [2], wherein the total amino acid content of the yeast extract is 0.75 part by mass or more relative to 100 parts by mass of the basal medium. [7] The basal medium for animal cells according to [1] or [2], wherein the yeast extract is a low-nucleic acid yeast extract. [8] The basal medium for animal cells according to [1] or [2], wherein the total nucleic acid content of the yeast extract is 2.0 parts by mass or less, relative to 100 parts by mass of the basal medium. [9] The basal medium for animal cells according to [1] or [2], wherein the yeast extract is a high-amino acid and low-nucleic acid yeast extract.

[10] The basal medium for animal cells according to [1] or [2], wherein the content of the yeast extract is 0.05 to 20.0 parts by mass, relative to 100 parts by mass of the basal medium.

[11] The basal medium for animal cells according to [1] or [2], further comprising a vitamin.

[12] The basal medium for animal cells according to [1] or [2], wherein the inorganic salt comprises calcium chloride.

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

[14] The culture method of

[13] , wherein the yeast extract is an extract of a high amino acid type yeast.

[15] The culture method of

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

[16] The culture method of

[13] , wherein the yeast extract is an extract of a yeast that is both high in amino acids and low in nucleic acids.

[17] Animal cells cultured by the culture method of

[13] .

[18] A processed food comprising the animal cells of

[17] .

[19] Use of a yeast extract in combination with amino acids, inorganic salts, and sugars in a basal medium for animal cells, wherein the amino acids are cystine and glutamine.

[0015] Yeast extract is a seasoning containing the amino acids, peptides, and nucleic acids found in yeast. By replacing part of the medium composition with yeast extract, the present invention reduces the amount of ingredients added to the composition compared to conventional commercially available basal medium compositions, thereby reducing the cost and environmental impact of animal cell culture.

[0016] Figure 1 shows cell images obtained in Example 1 to determine the minimum amino acid species required for addition. Figure 2 shows cell images obtained in Example 2 to determine the minimum amino acid species required for addition. Figure 3 shows the results of comparing the number of viable cells measured by absorbance at 450 nm when various yeast extracts were used in Example 4. From left to right, the results are for DMEM, PC (positive control), yeast extract A, yeast kiss B, yeast extract C, yeast kiss D, and N.C. (negative control) at each passage. Figure 4 shows the results of comparing the number of viable cells measured by absorbance at 450 nm when yeast extracts A and B were used, the amount of yeast extract added was increased to 2.0 g / L, and calcium chloride was added to the basal medium in Example 5. From left to right, the results are for PC (positive control), yeast extract A, yeast kiss B, and N.C. (negative control) at each passage. Figure 5 shows the results of measuring the viable cell count at the third passage when cells derived from various organs isolated from chickens were cultured using yeast extract A and yeast extracts A-B in Example 6. The results for liver, muscle, lung, kidney, and brain tissues are shown (from left to right) for PC (positive control), yeast extract A, and yeast extracts A-B. Figure 6 shows the results of examining the cell count when RL34 cells (cells derived from normal rat liver) were cultured using yeast extract A in Example 7. The vertical axis represents the value obtained when the cell count obtained when cultured using the positive control DMEM medium was set to 1.0. P1, P2, and P3 on the horizontal axis represent the results at the first, second, and third passages, respectively. Figure 7 shows the results of examining the cell count when MDBK cells (cells derived from normal bovine kidney) were cultured using yeast extract A in Example 7. The vertical axis represents the value obtained when the cell count obtained when cultured using the positive control DMEM medium was set to 1.0. P1, P2, and P3 on the horizontal axis represent the results at the first, second, and third passages, respectively. Figure 8 shows the results of examining the cell count when bovine muscle-derived cells were cultured using yeast extract A in Example 7. The vertical axis represents the value when the cell count when cultured using DMEM medium as a positive control is set to 1.0. P1, P2, and P3 on the horizontal axis represent the results at the first, second, and third passages, respectively.Figure 9 shows the results of three passages of MDBK cells using basal media supplemented with various amounts of amino acids in Example 8. The vertical axis of Figure 9 represents the number of viable cells. Figure 10 shows the results of three passages of MDBK cells (established cell line) (Figure 10A), RL34 cells (established cell line) (Figure 10B), and duck liver-derived cells (primary culture cells) (Figure 10C) using basal media containing L-cysteine ​​or L-cystine. The vertical axis of Figure 10 represents the number of viable cells after three passages.

[0017] The present invention includes, but is not limited to, the following embodiments. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in this specification, it means that the embodiment is not limited, i.e., is not limited.

[0018] 1. Composition for Basal Medium for Animal Cell Culture In one aspect, the present invention relates to a composition for a basal medium for animal cells.

[0019] The basal medium composition for animal cells comprises yeast extract, amino acids, inorganic salts, and sugars, and the amino acids in the composition comprise cystine and / or cysteine, and glutamine. In one embodiment, the basal medium composition for animal cells consists of yeast extract, amino acids, inorganic salts, and sugars, and the amino acids are cystine and / or cysteine, and glutamine.

[0020] The total amino acid content in the basal medium composition for animal cells is not particularly limited. Here, the "total amino acids" are components of the basal medium composition for animal cells, and are added to the basal medium composition as individual amino acids, and are distinguished from the amino acids contained in the yeast extract described below. "Total amino acids" includes cystine and / or cysteine ​​and glutamine, which are contained as essential components in the composition, as well as other types of amino acids added to the basal medium composition for animal cells.

[0021] Non-limiting examples of the total amino acid content in the composition are 5.0 parts by mass or less, 3.0 parts by mass or less, 2.0 parts by mass or less, 1.0 parts by mass or less, 0.5 parts by mass or less, 0.1 parts by mass or less, 0.08 parts by mass or less, or 0.07 parts by mass or less, when the basal medium composition is taken as 100. Non-limiting examples of the total amino acid content in the composition are 0.0005 parts by mass or more, 0.001 parts by mass or more, 0.002 parts by mass or more, 0.003 parts by mass or more, 0.004 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, when the basal medium composition is taken as 100.

[0022] Non-limiting examples of the total amino acid content in the composition are 0.0005 to 5.0 parts by mass, 0.001 to 5.0 parts by mass, 0.001 to 3.0 parts by mass, 0.002 to 3.0 parts by mass, 0.002 to 2.0 parts by mass, 0.002 to 1.0 parts by mass, 0.003 to 2.0 parts by mass, 0.005 to 1.0 parts by mass, 0.01 to 0.5 parts by mass, 0.03 to 0.1 parts by mass, 0.002 to 0.07 parts by mass, and 0.004 to 0.07 parts by mass, when the basal medium composition is taken as 100. Preferably, the amino acid content is 0.002 to 1.0 part by mass when the basal medium composition is taken as 100.

[0023] The ratio of cystine and / or cysteine ​​to glutamine is not particularly limited. The amounts of cystine and / or cysteine ​​and glutamine may be equal, or either may be present in a greater amount. Preferably, glutamine is present in a greater amount. In one embodiment, the amount of glutamine is 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, 8 times or more, or 10 times or more by mass than the amount of cystine and / or cysteine. In one embodiment, the amount of glutamine is about 12 times that of cystine and / or cysteine.

[0024] Cystine, cysteine, and glutamine may be in the L-form or the D-form. A portion of cystine, cysteine, and glutamine may be in the L-form, and the remaining portion may be in the D-form. In one embodiment, cystine, cysteine, and glutamine are in the L-form.

[0025] The phrase "the amino acids in the composition include cystine and / or cysteine, and glutamine" means that the main components of the amino acids are cystine and / or cysteine, and glutamine. In one embodiment, the ratio of cystine and / or cysteine, and glutamine to all amino acids in the composition is 50% or more. In one embodiment, the ratio of cystine and / or cysteine, and glutamine to all amino acids in the 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 ratio of cystine and / or cysteine, and glutamine to all amino acids in the composition is 100%. However, it is not limited to "a 100% proportion of cystine and / or cysteine, and glutamine," and it is presumed that the nutritional value and proliferation ability may be increased if a larger amount of other amino acids is added to the composition.

[0026] Non-limiting examples of the contents of cystine and / or cysteine, and glutamine among the amino acids in the composition are, when the basal medium composition is taken as 100, 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.

[0027] Non-limiting examples of the amino acids in the composition include when the basal medium composition is taken as 100, the content of cystine and / or cysteine, and glutamine is 0.00025 to 5.0 parts by mass, 0.0005 to 5.0 parts by mass, 0.0008 to 5.0 parts by mass, 0.0008 to 3.0 parts by mass, 0.001 to 5.0 parts by mass, 0.001 to 3 ... 0 parts by mass, 0.001 to 2.0 parts by mass, 0.001 to 1.0 parts by mass, 0.002 to 5.0 parts by mass, 0.002 to 3.0 parts by mass, 0.002 to 1.0 parts by mass, 0.0025 to 1.0 parts by mass, 0.003 to 2.0 parts by mass, 0.005 to 1.0 parts by mass, 0.01 to 0.5 parts by mass, 0.03 to 0.1 parts by mass, or 0.002 to 0.07 parts by mass. In one aspect, the content of cystine and / or cysteine, and glutamine among the amino acids in the composition is 0.001 to 1.0 part by mass, based on 100 parts of the basal medium composition. In one aspect, the content of cystine and / or cysteine, and glutamine among the amino acids in the composition is 0.002 to 1.0 part by mass, based on 100 parts of the basal medium composition.

[0028] Non-limiting examples of the contents of cystine and / or cysteine, and glutamine in the composition are 0.025 to 25.0 parts by mass, 0.05 to 25.0 parts by mass, 0.08 to 25.0 parts by mass, 0.08 to 15.0 parts by mass, 0.1 to 25.0 parts by mass, 0.1 to 15.0 parts by mass, 0.1 to 10.0 parts by mass, 0.1 to 5.0 parts by mass, 0.2 to 25.0 parts by mass, 0.2 to 15 parts by mass, 0.2 to 5.0 parts by mass, 0.25 to 5.0 parts by mass, 0.3 to 10.0 parts by mass, 0.5 to 5.0 parts by mass, 0.3 to 2.5 parts by mass, 0.4 to 2.0 parts by mass, and 0.1 to 3.5 parts by mass, based on the solid content of the basal medium composition. In one embodiment, the content of cystine and / or cysteine, and glutamine in the composition is 0.1 to 5.0 parts by mass per solid content of the basal medium composition. In another embodiment, the content of cystine and / or cysteine, and glutamine in the composition is 0.2 to 5.0 parts by mass per solid content of the basal medium composition.

[0029] Non-limiting examples of the content of cystine and / or cysteine ​​in the composition are 0.00001 to 0.2 parts by mass, 0.00005 to 0.15 parts by mass, 0.00008 to 0.1 parts by mass, 0.0001 to 0.1 parts by mass, 0.0001 to 0.08 parts by mass, 0.0001 to 0.04 parts by mass, 0.0001 to 0.02 parts by mass, 0.0001 to 0.01 parts by mass, 0.0002 to 0.1 parts by mass, 0.0002 to 0.08 parts by mass, 0.0002 to 0.05 parts by mass, 0.0002 to 0.02 parts by mass, 0.0002 to 0.01 parts by mass, 0.0002 to 0.005 parts by mass, or 0.0004 to 0.008 parts by mass, when the basal medium composition is taken as 100 parts. In one embodiment, the content of cystine and / or cysteine ​​in the composition is 0.0001 to 0.02 parts by mass when the basal medium composition is taken as 100 parts by mass.

[0030] Non-limiting examples of the content of cystine and / or cysteine ​​in the composition are 0.0005 to 10.0 parts by mass, 0.0025 to 7.5 parts by mass, 0.004 to 5.0 parts by mass, 0.005 to 5.0 parts by mass, 0.005 to 4.0 parts by mass, 0.005 to 2.0 parts by mass, 0.005 to 1.0 parts by mass, 0.005 to 0.5 parts by mass, 0.01 to 5.0 parts by mass, 0.01 to 4.0 parts by mass, 0.01 to 2.5 parts by mass, 0.01 to 1.0 parts by mass, 0.01 to 0.5 parts by mass, 0.01 to 0.30 parts by mass, and 0.02 to 0.40 parts by mass, relative to the solid content of the basal medium composition. In one embodiment, the content of cystine and / or cysteine ​​in the composition is 0.005 to 1.0 parts by mass per solid content of the basal medium composition.

[0031] Non-limiting examples of the content of glutamine among the amino acids in the composition are, when the basal medium composition is taken as 100, 0.00025 to 5.0 parts by mass, 0.0005 to 5.0 parts by mass, 0.0008 to 5.0 parts by mass, 0.0008 to 3.0 parts by mass, 0.001 to 5.0 parts by mass, 0.001 to 3.0 parts by mass, 0.001 to 2.0 parts by mass, 0.001 to 1.0 parts by mass, 0.002 to 5.0 parts by mass, 0.002 to 3.0 parts by mass, 0.002 to 1.0 parts by mass, 0.0025 to 1.0 parts by mass, 0.003 to 2.0 parts by mass, 0.005 to 1.0 parts by mass, 0.01 to 0.5 parts by mass, 0.03 to 0.1 parts by mass, and 0.002 to 0.06 parts by mass. In one embodiment, the content of glutamine among the amino acids in the composition is 0.001 to 1.0 part by mass when the basal medium composition is taken as 100. In another embodiment, the content of glutamine among the amino acids in the composition is 0.002 to 1.0 part by mass when the basal medium composition is taken as 100.

[0032] Non-limiting examples of the content of glutamine in the composition are 0.025 to 25.0 parts by mass, 0.05 to 25.0 parts by mass, 0.08 to 25.0 parts by mass, 0.08 to 15.0 parts by mass, 0.1 to 25.0 parts by mass, 0.1 to 15.0 parts by mass, 0.1 to 10.0 parts by mass, 0.1 to 5.0 parts by mass, 0.2 to 25.0 parts by mass, 0.2 to 15 parts by mass, 0.2 to 5.0 parts by mass, 0.25 to 5.0 parts by mass, 0.3 to 10.0 parts by mass, 0.5 to 5.0 parts by mass, 0.3 to 2.5 parts by mass, 0.4 to 2.0 parts by mass, and 0.1 to 3.2 parts by mass, based on the solid content of the basal medium composition. In one embodiment, the content of glutamine in the composition is 0.1 to 5.0 parts by mass per solid content of the basal medium composition. In another embodiment, the content of glutamine in the composition is 0.2 to 5.0 parts by mass per solid content of the basal medium composition.

[0033] The amino acids contained in the basal medium composition for animal cells are cystine and / or cysteine, and glutamine, and preferably do not contain amino acids other than cystine and / or cysteine, and glutamine. "Amino acids other than cystine and / or cysteine, and glutamine" are not particularly limited and include natural amino acids and unnatural amino acids. In one embodiment, the 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.

[0034] "Yeast extract" (also called "yeast extract") is a substance extracted from yeast cells. Generally, the main components are proteins and their decomposition products, peptides, amino acid and nucleic acid-related substances, minerals, and vitamins, and it is known to be used as a seasoning, livestock feed, health supplement, microbial culture medium, and quality improver for processed foods.

[0035] The amino acids contained in the yeast extract are constituents of the basal medium composition for animal cells, and are distinguished from the amino acids (i.e., including cystine and / or cysteine, and glutamine) that are added to the basal medium composition for animal cells as individual amino acids.

[0036] The type of yeast cells from which the yeast extract contained in the composition for a basal medium for animal cells is derived is not particularly limited. In one embodiment (i.e., without limitation), examples of yeast cells include cells of baker's yeast, brewer's yeast, sake yeast, and torula yeast. In one embodiment, baker's yeast (scientific name: Saccharomyces cerevisiae) is included.

[0037] Known yeast extracts include high-amino acid yeast extracts containing a large amount of amino acids, high-nucleic acid / high-amino acid yeast extracts, and high-nucleic acid yeast extracts, depending on the components contained. Yeast extracts containing a large amount of specific amino acids, organic acids, and / or nucleic acids are also known. For example, there are high-glutamic acid amino acid yeast extracts containing a high concentration of glutamic acid, particularly among amino acids; high-succinic acid yeast extracts containing a high concentration of succinic acid, particularly among organic acids; and high-nucleic acid yeast extracts containing a high concentration of inosinic acid and guanylic acid, particularly among nucleic acids.

[0038] The yeast extract may be, but is not limited to, a high amino acid yeast extract, a low nucleic acid yeast extract, or a high amino acid and low nucleic acid yeast extract.

[0039] In one embodiment, the high amino acid type yeast extract contains 30,000 mg or more, 35,000 mg or more, 40,000 mg or more, or 45,000 mg or more of total amino acids per 100 g of yeast extract.

[0040] When 100 g of yeast extract contains 30,000 mg of total amino acids, the amino acid content in the yeast extract is 30%. In Example 3 described below, yeast extract was added so that the final concentration in the culture medium was 0.25 g / L to 2.0 g / L. When the amino acid content in the yeast extract is 30%, the amino acid concentration in the culture medium is 0.075 g / L to 0.6 g / L (1000 g). In this case, the total amino acid content of the yeast extract is 0.75 parts by mass to 6.0 parts by mass, based on 100 parts of the basal medium composition.

[0041] Non-limiting examples of the total amino acid content of the yeast extract are 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, relative to 100 parts by mass of the basal medium composition. In one embodiment, the total amino acid content of the yeast extract is 0.75 parts by mass or more, relative to 100 parts by mass of the basal medium composition.

[0042] Non-limiting examples of the total amino acid content of the yeast extract are 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, when the basal medium composition is taken as 100 parts.

[0043] Non-limiting examples of the total amino acid content of the yeast extract are 0.30 to 20 parts by mass, 0.50 to 10 parts by mass, 0.60 to 8 parts by mass, or 0.75 to 6 parts by mass, when the basal medium composition is taken as 100 parts.

[0044] In one embodiment, the low nucleic acid type yeast extract contains no more than 10%, no more than 8%, no more than 5%, no more than 3%, no more than 2% nucleic acids in the yeast extract.

[0045] In Example 3 described below, yeast extract was added so that the final concentration in the culture medium was 0.25 g / L to 2.0 g / L. When the nucleic acid content in the yeast extract was 10%, the nucleic acid concentration in the culture medium was 0.025 g / L to 0.2 g / L (1000 g). In this case, the total nucleic acid content of the yeast extract was 0.25 parts by mass to 2.0 parts by mass, based on 100 parts of the basal medium composition.

[0046] Non-limiting examples of the total nucleic acid content of the yeast extract are 3.0 parts by mass or less, 2.0 parts by mass or less, 1.5 parts by mass or less, 1.0 parts by mass or less, 0.7 parts by mass or less, 0.5 parts by mass or less, and 0.3 parts by mass or less, relative to 100 parts by mass of the basal medium composition. In one embodiment, the total nucleic acid content of the yeast extract is 2.0 parts by mass or less, relative to 100 parts by mass of the basal medium composition.

[0047] Non-limiting examples of the total nucleic acid content of the yeast extract are 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, relative to 100 parts by mass of the basal medium composition. In one embodiment, the total nucleic acid content of the yeast extract is 0.25 parts by mass or more, relative to 100 parts by mass of the basal medium composition.

[0048] Non-limiting examples of the total nucleic acid content of the yeast extract are 0.05 to 2.0 parts by mass, 0.10 to 2.0 parts by mass, 0.20 to 2.0 parts by mass, or 0.25 to 2.0 parts by mass, when the basal medium composition is taken as 100 parts.

[0049] The yeast extract contained in the composition for a basal animal culture medium may be one type or two or more types.

[0050] Without limitation, the yeast extract may be any one of yeast extracts A, B, C, or D described in the Examples below, or a mixture thereof. In one embodiment, the yeast extract may be yeast extract A or B described in the Examples below, or a mixture thereof. In one embodiment, the yeast extract may be yeast extract A described in the Examples below. Specific examples include yeast extract YK-21NL, Himax PR, YP21-CM, and yeast extract 21-NYP (all manufactured by Fuji Foods Industry Co., Ltd.).

[0051] The content of the yeast extract in the basal medium composition for animal cells is not particularly limited. The content of the yeast extract is desirably a concentration that is effective in promoting the proliferation of animal cells, but does not inhibit proliferation (due to excessive concentration). Non-limiting examples of the yeast extract content in the basal medium composition for animal cells, based on 100 parts of the basal medium composition, include 0.01 to 25.0 parts by mass, 0.05 to 20.0 parts by mass, 0.10 to 15.0 parts by mass, 0.20 to 10.0 parts by mass, 0.25 to 5.0 parts by mass, and 0.25 g to 2.0 parts by mass. In one embodiment, the yeast extract content in the basal medium composition for animal cells is 0.05 to 20.0 parts by mass, based on 100 parts of the basal medium composition.

[0052] The yeast extract may be used in combination with the yeast extract, or an extract derived from a natural product other than the yeast extract may be used as a component of the basal medium composition for animal cells. Examples of the extract derived from a natural product include, but are not limited to, extracts derived from grains 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 fungal bodies and mycelia.

[0053] In one embodiment, the extract derived from a natural product other than yeast extract does not include soy hydrolysate.

[0054] The type of inorganic salt contained in the basal medium composition for animal cells is not particularly limited. Examples include, but are not limited to, salts of calcium, iron, potassium, magnesium, sodium, zinc, and the like. Examples of salts include, but are not limited to, chlorides, sulfates, phosphates, chlorides, bicarbonates, nitrates, and the like. Examples of inorganic salts include, but are not limited to, calcium chloride, iron chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate, iron sulfate, iron nitrate, magnesium chloride, zinc sulfate, and the like. In one embodiment, calcium chloride is contained as the inorganic salt. In another embodiment, the basal medium composition for animal cells may contain two or more types of inorganic salts.

[0055] In one embodiment, the basal medium composition for animal cells comprises two or more inorganic salts selected from the group consisting of calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate. In another embodiment, the basal medium composition for animal cells comprises calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate.

[0056] The amount of the inorganic salt contained in the composition for a basal medium for animal cells is not particularly limited, and a concentration appropriate for culturing animal cells can be appropriately selected depending on the type of inorganic salt.

[0057] "Sugars" are the initial oxidation products of polyhydric alcohols and have one formyl group (-CHO) or one carbonyl group (>C=O). Sugars with a formyl group are classified as aldoses, while sugars with a carbonyl group are classified as ketoses. Sugars are found in large amounts in plant tissues, honey, and fruits, and glucose in particular is a source of energy for living organisms.

[0058] A molecule formed by two monosaccharide molecules bound together through a glycosidic bond is called a disaccharide, a molecule formed by three monosaccharide molecules bound together is called a trisaccharide, and a molecule formed by four monosaccharide molecules is called a tetrasaccharide. A molecule formed by the bond of 2-20 monosaccharide molecules is called an oligosaccharide. A molecule formed by the polymerisation of many monosaccharides through glycosidic bonds is called a polysaccharide. A sugar in which the hydroxyl group is replaced with hydrogen is called a deoxysugar, a sugar in which the terminal carbon of an aldose is replaced with a carboxyl group is called a uronic acid, a sugar in which the hydroxyl group is replaced with an amino group is called an amino sugar, and a sugar alcohol in which a ketone group or aldehyde group is reduced to an alcohol.

[0059] Known examples of sugars include the monosaccharides glucose (grape sugar), mannose, fructose (fruit sugar), galactose, and xylose. Known disaccharides consisting of two glucose molecules include trehalose, isotrehalose, maltose (malt sugar), cellobiose, and isomaltose. Other known disaccharides include lactose (a 1,4-galactosidic bond between β-galactose and β-glucose) and sucrose (a 1,2-glycosidic bond between α-glucose and β-fructofuranose (fructose)).

[0060] The type of sugar contained in the basal medium composition for animal cells is not particularly limited. Examples include, but are not limited to, glucose (dextrose), fructose, trehalose, galactose, sucrose, and maltose. In one embodiment, the sugar contained in the basal medium composition for animal cells is glucose (dextrose). In another embodiment, the basal medium composition for animal cells may contain two or more types of sugars.

[0061] The amount of the sugar contained in the composition for a basal medium for animal cells is not particularly limited, and a concentration appropriate for culturing animal cells can be appropriately selected depending on the type of sugar.

[0062] In one embodiment, the composition for a basal medium for animal cells may further contain a vitamin.

[0063] "Vitamin" is a general term for organic compounds other than carbohydrates, proteins, and lipids that are necessary in trace amounts for the survival and growth of living organisms, but cannot be synthesized in sufficient amounts within the organism's body. Vitamins are classified by function, not by substance name. For example, vitamin A is made up of retinal, retinol, etc. Vitamins are classified into fat-soluble vitamins and water-soluble vitamins. Below are examples of vitamins for humans.

[0064] Fat-soluble vitamins Vitamin A: Retinol, β-carotene, α-carotene, β-cryptoxanthin, etc. Vitamin D: Ergocalciferol, cholecalciferol Vitamin E: Tocopherol, tocotrienol Vitamin K: Two naphthoquinone derivatives, phylloquinone and menaquinone Water-soluble vitamins Vitamin B group Vitamin B １ : Thiamine Vitamin B ２ : Riboflavin, also known as vitamin G, vitamin B ３ Niacin, also known as vitamin PP, vitamin B ５ : Pantothenic acid Vitamin B ６ : Pyridoxal, pyridoxamine, pyridoxine Vitamin B ７ Biotin. Also known as Vitamin Bw or Vitamin H. Vitamin B ９ : Folic acid. Also known as vitamin Bc and vitamin M. Vitamin B １２ : Cyanocobalamin, methylcobalamin, hydroxocobalamin Vitamin C: Ascorbic acid

[0065] Substances that do not fit the definition of vitamins above but have effects similar to vitamins are sometimes called "vitamin-like substances." Vitamin-like substances include those that were historically mistakenly considered vitamins, or those that are no longer considered vitamins due to changes in the definition. Examples of vitamin-like substances are listed below.

[0066] Vitamin B ４ : Adenine Vitamin B８ : Ergadenylic acid (adenylic acid) Vitamin B １０ : A mixture of folic acid and various B vitamins. Also called vitamin R. Vitamin B １１ : Folic acid analogue. Vitamin B １３ : Orotic acid Vitamin B １４ : Folic acid or lipoic acid, etc. Vitamin B １５ : Pangamic acid Vitamin B １６ : Dimethylglycine Vitamin B １７ : Amygdalin Vitamin B Ｈ : Inositol Vitamin B Ｐ : Choline Vitamin B Ｔ : Carnitine Vitamin B Ｘ : Para-aminobenzoic acid (partial structure of folic acid, also known as PABA) Vitamin F: Essential fatty acids such as linoleic acid Vitamin I: Extract of rice bran Vitamin J: Catechol, flavin or choline Vitamin L １ : Anthranilic acid Vitamin L ２ : Adenylthiomethylpentose Vitamin N: Thioctic acid (α-lipoic acid) Vitamin O: Carnitine Vitamin P: Flavonoids such as quercetin, hesperidin, rutin, and eriocitrin Vitamin Q: Ubiquinone Vitamin S: Salicylic acid Vitamin T: Tegotin Vitamin U: Methylmethionine sulfonium chloride (also known as "cabegin") Vitamin V: Nicotinamide adenine dinucleotide

[0067] The type of vitamin contained in the composition for a basal medium for animal cells is not particularly limited. In this specification, unless otherwise specified, the term "vitamin" includes the above-mentioned vitamin-like substances.

[0068] The vitamins contained in the basal medium composition for animal cells are, without limitation, one or more vitamins selected from the group consisting of pantothenate (e.g., calcium pantothenate), folic acid, pyridoxine or a salt thereof (e.g., pyridoxyl 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 basal medium composition for animal cells contains all of pantothenate (e.g., calcium pantothenate), folic acid, pyridoxine or a salt thereof (e.g., pyridoxyl hydrochloride), riboflavin, thiamine or a salt thereof (e.g., thiamine hydrochloride), choline (e.g., glycerophosphatidylcholine), inositol (e.g., myo-inositol), and nicotinamide (vitamin V).

[0069] The amount of the vitamin contained in the composition for a basal medium for animal cells is not particularly limited, and a concentration appropriate for culturing animal cells can be appropriately selected depending on the type of vitamin.

[0070] The basal medium composition for animal cells may contain other components in addition to yeast extract, amino acids, inorganic salts, vitamins, and sugars. The other components may be any components that can be used in a basal medium composition for animal cells, including, for example, a pH indicator (phenol red), glutathione, fatty acids such as linolenic acid, and thymidine.

[0071] A "basal medium for animal cells" is a medium that serves as a base for a culture medium for culturing, growing, and / or differentiating animal cells. It is typically used with the addition of serum or other ingredients, without limitation. In this specification, "culture" also includes the meaning of growth unless otherwise specified. In this specification, a "basal medium composition for animal cells" is a composition that serves as a base for a basal medium for culturing animal cells. The basal medium composition for animal cells may be a liquid or solid composition. For example, a basal medium composition for animal cells may be a liquid composition and used as a basal medium for animal cells as is. Alternatively, the composition may be in the form of a paste from which water has been removed, and stored or sold by adjusting the composition to an appropriate water activity. An appropriate amount of water may be added to use the composition as a basal medium for animal cells. Alternatively, the solid composition may be stored or sold as a powdered raw material that has been dried, and then dissolved in a solvent (e.g., water) before use and used as a basal medium for animal cells. In this specification, the terms "basal medium for animal cells" and "basal medium" are sometimes used synonymously with "basal medium composition for animal cells."

[0072] The type of animal from which the animal cells are derived is not particularly limited. In one embodiment, the animal cells are cells of a vertebrate such as a mammal. The vertebrate may be any of mammals, reptiles, birds, and amphibians, and includes, for example, humans, cows, pigs, rabbits, chickens, sheep, and goats. In one embodiment, the vertebrate is a mammal other than a human. In one embodiment, the animal cells are cells derived from animals that lay eggs with shells (oviparous), such as birds and reptiles. In one embodiment, the animal cells are chicken cells.

[0073] The type of animal cell is not particularly limited, and may be, for example, any of somatic cells derived from adults, embryos, cultured cells established from fertilized eggs, etc. Non-limiting examples include chicken embryos, preferably embryos from day 0 to day 21, day 2 to day 19, day 4 to day 17, and day 6 to day 15.

[0074] The tissue from which the animal cells are derived is not particularly limited, and includes, but is not limited to, muscle-derived cells, skin-derived cells, intestinal-derived cells, heart-derived cells, brain-derived cells, stomach-derived cells, embryonic membrane-derived cells, liver-derived cells, kidney-derived cells, and lung-derived cells.

[0075] In the following embodiment, the amino acids are cystine and / or cysteine, and glutamine, and the amino acids cystine and / or cysteine, and glutamine are the main components. In one embodiment, the composition does not contain any amino acids other than cystine and / or cysteine, and glutamine, but is not limited to this. Although cystine and / or cysteine, and glutamine are essential elements of the basal medium composition for animal cells, it is expected that the growth of the animal cells may be nutritionally promoted by combining them with other amino acids.

[0076] In one aspect, the present invention relates to the use of a yeast extract in combination with amino acids, inorganic salts, and sugars in a composition for a basal medium for animal cells, wherein the amino acids are cystine and / or cysteine, and glutamine.

[0077] In one aspect, the present invention relates to a combination of yeast extract, amino acids, inorganic salts, and sugars for use in a basal medium composition for animal cells, wherein the amino acids are cystine and / or cysteine, and glutamine.

[0078] In one aspect, the present invention relates to a kit for producing a basal medium composition for animal cells, comprising yeast extract, amino acids, inorganic salts, and sugars, wherein the amino acids are cystine and / or cysteine, and glutamine. In the kit, the yeast extract, amino acids, inorganic salts, and sugars may be present individually or in a mixture of two or more components until the production of the basal medium composition for animal cells is performed.

[0079] 2. Method for Culturing Animal Cells In one aspect, the present invention relates to a method for culturing animal cells.

[0080] Non-limiting examples of the culture method include culturing animal cells in a basal animal cell medium containing yeast extract, amino acids, inorganic salts, and sugars, where the amino acids include L-cystine and / or L-cysteine, and L-glutamine.

[0081] The culture conditions in the method for culturing animal cells are not particularly limited, and appropriate culture conditions can be adopted depending on the animal species, tissue, etc. from which the animal cells are derived.

[0082] In one embodiment, the yeast extract is an extract of a high amino acid type yeast.

[0083] In one embodiment, the yeast extract is an extract of a low nucleic acid type yeast.

[0084] In one embodiment, the yeast extract is an extract of yeast that is high in amino acids and low in nucleic acids.

[0085] In one embodiment, the basal animal cell medium used in the method for culturing animal cells may contain vitamins.

[0086] The meanings of "yeast extract," "amino acids," "inorganic salts," "vitamins," "sugars," and "basal medium for animal cells" are as described in "1. Composition for basal medium for animal cell culture."

[0087] The matters described in "1. Composition for basal medium for animal cell culture" are also incorporated into the culture method of this section unless otherwise inconsistent.

[0088] In one aspect, the present invention relates to a medium for use in a method for culturing animal cells, comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids include L-cystine and / or L-cysteine, and L-glutamine.

[0089] In one aspect, the present invention relates to the use of a medium comprising yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids include L-cystine and / or L-cysteine, and L-glutamine, in a method for culturing animal cells.

[0090] 3. Animal Cells and Processed Products In one aspect, the present invention includes animal cells cultured by the animal cell culture method of the present invention.

[0091] In one aspect, the present invention relates to a processed food comprising the animal cells.

[0092] The meanings of "animal cell culture method," "animal cells," etc. are as described in "1. Composition for basal medium for animal cell culture" and "2. Animal cell culture method."

[0093] The matters described in "1. Composition for basal medium for animal cell culture" and "2. Method for culturing animal cells" are also applicable to the animal cells and processed foods in this section unless there is a particular contradiction.

[0094] The animal cells and processed foods cultured by the animal cell culture method of the present invention can be produced with less environmental impact than when conventional commercially available basal medium compositions are used. As a result, animal cells and processed foods can be obtained at low cost and with less environmental impact. In addition, they have a more complex flavor that cannot be obtained by animal cell culture methods, and are characterized by the unique meat flavor of original animal tissue.

[0095] "Processed foods" include, for example, meat containing cultured animal cells or made from cultured animal cells (cultured meat). Cultured meat can be cooked and used as an ingredient in the same way as regular meat, such as meat from cows, pigs, rabbits, chickens, sheep, or goats. The "food" in "processed foods" can mean both ingredients before cooking and food (meals) after cooking. The "processing" in "processed foods" does not simply mean cooking ingredients to create meals, but also refers to the entire processing of ingredients, such as pre-processing ingredients to produce more highly processed ingredients, or processing ingredients to provide new ingredients with adjusted taste, physical properties, etc.

[0096] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0097] Example 1: Investigation of the minimum amino acid species that need to be added In this example, when yeast extract is used as an amino acid source in culturing animal cells, it was investigated whether cells can grow using yeast extract alone without the addition of additional amino acids, and if not, which amino acids are the minimum amino acids that are needed.

[0098] (Method) The components contained in Dulbecco's modified Eagle's medium (DMEM) except for amino acids were dissolved in ion-exchanged water to prepare AA-IMEM buffer (Table 1).

[0099] Table 1 A. A. -IMEM Buffer composition

[0100]

[0101] A. Seven types of yeast extracts shown below (B, D, E, F, H, I, and J) were further added to the A.-IMEM buffer so that the final concentration in the medium was 1 g / L.

[0102] Table 2: Yeast extracts added in Example 1

[0103]

[0104] Various amino acids required for cell growth in DMEM medium were added to "A.A.-IMEM Buffer + yeast extract" according to conditions 1 to 16 in Table 3, and a cell culture test was conducted. This allowed us to determine the essential amino acids that must be added for cell growth when yeast extract is used as a culture material. The amount of each amino acid added was the same as the amount contained in the DMEM medium composition. Furthermore, a PC (positive control) with the same amino acid composition as DMEM medium was used, while condition 19 and condition 20 were used with the same amino acid composition but with 1 / 8 the amount added. All reagents used in the experiment were food additive grade.

[0105] Table 3. Amino acid conditions of test medium

[0106]

[0107] A. A. A.-IMEM Buffer + yeast extract, and the amino acids listed in Table 3 were further supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin (PSA) to conduct a cell culture test.

[0108] For the cell culture test, mouse-derived myoblast cell line C2C12 was used. C2C12 cells were cultured in a 12-well plate at 5 × 10 ４ Cells were seeded at 1000 cells / well and incubated at 37°C in CO ２ The cells were cultured in the medium for each condition in an incubator for 3 days. After culturing under each condition, the cells were observed for adhesion and proliferation. Furthermore, images of the cells after culturing were taken to confirm the state of cell growth.

[0109] (Results) The results of the cell images are shown in Figure 1. From the cell images in Figure 1, the percentage of cells occupying the bottom surface of the culture vessel was marked with ◯, 10-50% with △, and no cell adhesion was observed with ×. The results are shown in Table 4.

[0110] Table 4. Cell proliferation scores under each condition

[0111]

[0112] As shown in Figure 1 and Table 4, when all amino acids were added (P.C.) or when 1 / 8 of the amino acids were added (condition 19), cell adhesion and subsequent proliferation were observed, whereas when 1 / 32 of the amino acids were added (condition 20) or when no amino acids were added (condition 1), cell adhesion was not observed. This demonstrates that yeast extract alone (final concentration in the medium: 1 g / L) does not provide enough amino acids necessary for cell adhesion and proliferation, and that additional amino acids must be added.

[0113] The amino acids required in DEME medium were each added individually to examine which amino acids were required in addition to yeast extract. As a result, cell adhesion was not observed when most amino acids were added alone, but cell adhesion was observed only under condition 15, in which L-cystine was added. This demonstrates that adding L-cystine to yeast extract improves cell adhesion.

[0114] Furthermore, when 1 / 8 or more of all amino acids were added (condition 19, P.C.), cells proliferated and adhered, whereas the percentage of adherent cells was significantly reduced under condition 18, in which only L-glutamine was omitted, demonstrating that L-glutamine is necessary for cell proliferation. On the other hand, under condition 16, in which only L-glutamine was added, no cell adhesion was observed, indicating that L-glutamine alone is insufficient and that L-cystine is also required. These results demonstrate that the minimum amino acids required for a basal medium are L-cystine and L-glutamine.

[0115] Furthermore, cell adhesion and proliferation were confirmed under condition 19, whereas almost no cells adhered under condition 20, suggesting that at least 6.13 mg / L of L-cystine and 74.5 mg / L of L-glutamine are required.

[0116] Example 2: Culture Tests in Multiple Cell Types Example 1 demonstrated that the addition of L-cystine and L-glutamine is necessary for the culture of C2C12 cells. In this example, we investigated whether these amino acids are essential not only for C2C12 cells, a mouse-derived myoblast cell line, but also for other established cell lines. Cell culture tests were performed in the same manner as in Example 1 using MDBK cells (a bovine kidney-derived cell line) and HDFa cells (an adult human dermal fibroblast cell line).

[0117] (Method) The DMEM medium composition, excluding amino acids, was dissolved in ion-exchanged water to prepare A.A.-IMEM Buffer (Table 1). Equal amounts of each yeast extract used in Example 1 were blended to prepare a yeast extract mix. This yeast extract mix was then added to the medium to a final concentration of 1 g / L. L-cystine or L-cystine and L-glutamine were added as shown in Table 5, and the cell growth of each cell type was confirmed. Cell culture tests were performed using A.A.-IMEM Buffer + yeast extract, the amino acids listed in Table 5, and 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin (PSA).

[0118] Table 5. Amino acid conditions of test medium

[0119]

[0120] MDBK cells and HDFa cells were each cultured at 5 × 10 ４ The cells were seeded at 1 cell / well and cultured in a CO2 incubator at 37°C. The MDBK cells were photographed on day 5 of culture, and the HDFa cells were photographed on day 4 of culture, to confirm the state of cell adhesion.

[0121] (Results) The results of the cell images are shown in Figure 2. From the cell images in Figure 2, the percentage of cells occupying the bottom surface of the culture vessel was marked with ◯, 10-50% with △, and no cell adhesion was observed with ×. The results are shown in Table 6.

[0122] Table 6. Cell proliferation scores under each condition

[0123]

[0124] As shown in Figure 2 and Table 6, no cell adhesion was observed in either cell type under condition 1, in which only yeast extract mix was added without adding any amino acids, whereas cell adhesion was observed under condition 2, in which L-cystine was added. Furthermore, cell proliferation was confirmed under condition 3, in which L-cystine and L-glutamine were added.

[0125] These results confirmed that L-cystine and L-glutamine significantly contribute to cell adhesion and proliferation. It was revealed that the inclusion of L-cystine, L-glutamine, and yeast extract in the medium made it possible to culture at least the three types of established cell lines examined in Examples 1 and 2.

[0126] Example 3: Investigation of yeast extracts to be added - Tests to confirm the suitability of various yeast extracts for cell proliferation In this example, in order to select the optimum yeast extract to add to the medium, tests to confirm the suitability of various yeast extracts for cell proliferation were carried out using several yeast extracts with different characteristics.

[0127] (Method) A basal medium having the composition shown in Table 7 was used.

[0128] Table 7. Test basal medium

[0129]

[0130] Eight types of yeast extracts A to H in Table 8 were added to the medium so that the final concentrations in the medium were 0.25 g / L, 0.5 g / L, 1.0 g / L, and 2.0 g / L, respectively. All eight types of yeast extracts were derived from the genus Saccharomyces and had the characteristics shown in Table 8.

[0131] Table 8. Types of yeast extract used

[0132]

[0133] Duck liver-derived cells, which are primary cultured cells, were used for the test. After being extracted from the animal, the duck liver-derived cells were cultured for up to three passages in a basal medium with the same composition as DMEM medium, supplemented with 10% serum and 1% PSA. Duck liver-derived cells were cultured in a 96-well plate at 1.5 x 10 ４ The cells were seeded at 1 cell / well, and 10% serum and 1% PSA were added to each medium, followed by a cell culture test.

[0134] Cell proliferation was monitored using an Incucyte SX5 (Sartorius) and the proportion of cells in the observed image was recorded as the cell area ratio over time for each medium. The recorded data was fitted with a logistic curve, and the cell proliferation midpoint (Exponential Growth 50%: EG50) was calculated and the cell proliferation rates were compared. Cells that took longer than 100 hours to reach EG50 were determined to have the ability to inhibit cell proliferation.

[0135] Furthermore, DMEM medium (Fujifilm) was used for PC, and the basal medium shown in Table 6 without yeast extract was used for NC.

[0136] (Results) Table 9 shows the results of the EG50 arrival time of cells cultured in each medium. Comparing the EG50 values ​​in Table 9, it was found that cell proliferation ability decreased when yeast extract B, yeast extract E, and yeast extract F were added at high concentrations of 2.0 g / L and 1.0 g / L. However, these growth inhibition effects decreased at lower concentrations. Furthermore, for yeast extract F, yeast extract H, and yeast extract G, which does not have cell proliferation inhibition ability at high concentrations, the EG50 arrival time was 100 hours or more even at additions of 0.5 g / L or less, resulting in a decrease in cell proliferation rate. These results demonstrate that these yeast extracts inhibit cell proliferation even at low concentrations.

[0137] Table 9. Time (h) for cells cultured in each medium to reach EG50

[0138]

[0139] Example 4: Investigation of yeast extracts to be added - Selection of optimal yeast extract In this example, an experiment was conducted to select the yeast extract most suitable for cell growth. The yeast extracts used for the selection were yeast extracts A, B, C, and D, excluding the yeast extract found to have cell growth inhibitory activity at low concentrations in Example 3.

[0140] (Method) Duck liver-derived cells, which are primary cultured cells, were used in the experiment. After being removed from the animal, the duck liver-derived cells were cultured for the 5th to 6th passages in a basal medium with the same composition as DMEM medium, but with 10% serum and 1% PSA added. The cells were repeatedly subcultured to examine whether they could proliferate.

[0141] Each yeast extract was added to the test basal medium shown in Table 7 so that the final concentration in the medium was 1.0 g / L, and 10% serum and 1% PSA were further added to prepare a complete medium.

[0142] Duck liver cells were plated in a 60 mm dish at 1.5 × 10 ５ At the time of subculture, a portion of the cells was transferred to a 96-well plate at 5 × 10 ３Cells were seeded at 100 cells / well, and the viable cell counts were compared by measuring absorbance at 450 nm using Cell Counting Kit-8 (Dojindo Laboratories). Culture was continued up to the third passage, and the viable cell counts when cultured with each yeast extract were compared based on the absorbance values.

[0143] (Results) In N.C. (negative control) without yeast extract, cells did not grow and could no longer be cultured after the second passage. On the other hand, it was revealed that duck liver-derived cells grew even after subculture in all media containing yeast extract. Furthermore, it was confirmed that cells grew more rapidly in media containing yeast extract A, yeast extract B, and yeast extract C than in media containing other yeast extracts (Figure 3).

[0144] Example 5 Optimization of Yeast Extract-Supplemented Medium In this example, the optimum medium composition was investigated using yeast extracts A and B, which showed particularly good cell growth in Example 5.

[0145] (Method) Duck liver-derived cells, which are primary cultured cells, were used in the experiment. After being removed from the animal, the duck liver-derived cells were cultured for the 5th to 6th passages in a basal medium with the same composition as DMEM medium, but with 10% serum and 1% PSA added. The cells were repeatedly subcultured to examine whether they could proliferate.

[0146] Complete media were prepared by adding 2.0 g / L of each yeast extract, 10% serum, and 1% PSA to the test basal media shown in Table 10. In this test, calcium chloride was added to the basal medium composition shown in Table 7 of Example 4 in an amount equal to the amount of calcium chloride contained in DMEM medium (Table 10).

[0147] Table 10. Test basal medium

[0148]

[0149] Duck liver cells were plated in a 60 mm dish at 1.5 × 10 ５ At the time of subculture, a portion of the cells was transferred to a 96-well plate at 5 × 10 ３Cells were seeded at 100 cells / well, and the number of viable cells was compared by measuring absorbance at 450 nm using Cell Counting Kit-8 (Dojindo Laboratories). Culture was continued up to the fifth passage, and the number of viable cells cultured in each yeast extract was compared as absorbance values.

[0150] The P.C. (positive control) used a medium with the same composition as the DMEM medium, and the N.C. (negative control) used only the test basal medium with the composition shown in Table 10, without adding yeast extract.

[0151] (Results) The results are shown in Figure 4. It was revealed that culturability improved by increasing the amount of yeast extract to 2.0 g / L and adding calcium chloride to the basal medium. N.C. cells stopped growing at the third passage and could no longer be cultured, but it was confirmed that the culture medium containing yeast extract maintained approximately 70% or more of the culturability of P.C. at the fifth passage.

[0152] Example 6 Versatility Confirmation Test 1 of Yeast Extract-Supplemented Medium In this example, yeast extract A, which showed particularly good cell growth in Example 5, and yeast extract A-B, a blend of equal amounts of A and B, were used to carry out culture tests on cells derived from various chicken organs, and a versatility confirmation test was carried out for these yeast extracts.

[0153] (Method) Experiments were performed using primary cultured cells derived from chicken liver, muscle, lung, kidney, and brain. Cells derived from various chicken organs were isolated from the body and cultured in the appropriate media. The yeast extract-supplemented medium was prepared by adding vitamins contained in DMEM medium (calcium pantothenate, folic acid, pyridoxyl hydrochloride, riboflavin, thiamine hydrochloride, glycerophosphatidylcholine, myo-inositol, and nicotinamide in Table 1) to the test basal medium shown in Table 10 of Example 5 in the same amounts as the DMEM medium, and adding 2.0 g / L of yeast extract A. A basal medium with the same composition as DMEM medium was also used as a positive control. These media were supplemented with 10% serum and 1% PSA for the tests.

[0154] Cells derived from various organs extracted from chickens were seeded in yeast extract-supplemented medium and positive control medium, respectively, and subcultured every 3 to 4 days. The number of cells seeded at the third subculture was 3 x 10 ５ The cells were seeded into a 6-well plate at 100 cells / well.

[0155] (Results) The results are shown in Figure 5. Cells derived from various chicken organs cultured in a medium supplemented with yeast extract A showed growth comparable to that of the positive control. This demonstrates that yeast extract-supplemented media can maintain good culturability for cells derived from various organs. Furthermore, the culturability of lung, kidney, and brain cells was slightly reduced in the medium supplemented with yeast extracts A and B, but the culturability of liver and muscle cells was comparable to that of PC and yeast extract A-supplemented media.

[0156] Example 7 Verification Test 2 of Yeast Extract-Supplemented Medium In this example, yeast extract A, which had excellent proliferation properties in Example 6, was used to carry out a culture test on established cell lines and primary cultured cells of different origins, and the culturability was confirmed.

[0157] The experiments used the established cell line RL34 (cells derived from normal rat liver), MDBK (cells derived from normal bovine kidney), and primary cultured bovine muscle cells. The yeast extract-supplemented medium was prepared by adding the vitamins contained in DMEM medium (calcium pantothenate, folic acid, pyridoxyl hydrochloride, riboflavin, thiamine hydrochloride, glycerophosphatidylcholine, myo-inositol, and nicotinamide in Table 1) to the basal medium of the composition shown in Table 10 of Example 5 in the same amount as the DMEM medium composition, and adding 2.0 g / L of yeast extract A. DMEM medium was used as a positive control. These media were supplemented with 10% heat-inactivated FBS and 1% PSA before use.

[0158] (Method) Each cell type was cultured in DMEM medium and placed in a 6-well plate at 3 x 10 RL34 cells. ５ cells, MDBK cells were 2 × 10 ５ cells, bovine muscle-derived cells were 3 × 10 ５The cells were seeded in the following amounts and cultured in each medium. Subculture was performed every 3 to 4 days, and the cell number was counted.

[0159] (Results) The results are shown in Figures 6 to 8. It was confirmed that for all cells, the cells cultured in the medium supplemented with yeast extract A maintained 70% or more of the culturability compared to the positive control DMEM medium.

[0160] Example 8 Investigation of the Lower Limit Range of Amino Acid Concentration Added to Basal Medium In this example, the concentration range of amino acids (L-glutamine, L-cystine) added to the basal medium, particularly the minimum required concentration, was investigated.

[0161] (Method) MDBK cells cultured in DMEM medium were placed in a 12-well plate at 1 × 10 ５ The cells were seeded at 100 cells / well, and 2 ml of medium was added to each well for culturing. The cells were subcultured every 3 to 4 days, and the number of viable cells was measured at the end of the third subculture.

[0162] Each medium used in the test contained 10% heat-treated FBS and 1% PSA.

[0163] Cells used: MDBK cells (established cell line) Number of cells before culture: 1 x 10 ５ Cells Amino acid addition concentration range: 0.002 to 0.063 mass% 0.063 mass% was set as 1, and the amount of amino acid added was reduced by half from there until the lowest amount was 0.002 mass%. The composition of the basal medium is shown in the table below.

[0164]

[0165] The results of cell proliferation when cultured in the above basal medium are shown in Figure 9. The horizontal axis of Figure 9 represents parts by mass (w / v, %) of amino acids (L-glutamine, L-cystine) contained in the basal medium, with the basal medium being taken as 100. The vertical axis of Figure 9 represents the number of viable cells at the end of the third subculture.

[0166] (Results) At the second passage, MDBK cells were confirmed to grow even when the amount of amino acid added was reduced to 0.002% by mass. Furthermore, at the third passage, cell growth was not observed at 0.002% by mass, but cell growth was still possible. Furthermore, cell growth was confirmed at 0.004% by mass or higher, even at the third passage.

[0167] From the above results, it was found that the lower limit range of the concentration of L-cystine and L-glutamine contents was 0.002% by mass when the basal medium was taken as 100.

[0168] Example 9 Confirmation of Cultivability When L-cystine is Replaced with L-cysteine ​​In this example, the cultivability when L-cystine is replaced with L-cysteine ​​was confirmed.

[0169] In this example, when L-cysteine ​​hydrochloride monohydrate, which has a higher solubility than L-cystine, was used as an amino acid to be added to a basal medium, its cultivability was examined in comparison with that of L-cystine.

[0170] (Method) Cells used: MDBK cells (established cell line), RL34 cells (established cell line), duck liver-derived cells (primary cultured cells) Number of cells before culture: MDBK and RL34 cells = 1 x 10 ５ Cells / well, duck liver-derived cells = 1 x 10 ６ Cells / 10 cm dish Amount of L-cysteine ​​hydrochloride monohydrate added: Since L-cystine is a dimer of L-cysteine, it was added in an amount twice the number of moles of L-cystine.

[0171] The composition of the basal medium is shown in the table below.

[0172]

[0173] MDBK cells and RL34 cells were subcultured in complete medium containing the above basal medium plus 1% PSA and 10% FBS. Duck liver-derived cells were subcultured in complete medium containing the above basal medium plus 1% PSA and 10% serum. Both were subcultured every 3-4 days, up to the third subculture.

[0174] (Results) The results at the end of the third subculture are shown in Figure 10. The vertical axis in Figure 10 represents the number of viable cells. Culture tests were performed using established cell lines MDBK cells (established cell lines) and RL34 cells, as well as primary cultured duck liver cells. In all cases, almost no difference in culturability was observed when L-cysteine ​​hydrochloride monohydrate was substituted for L-cystine.

[0175] These results demonstrate that the addition of either L-cystine or L-cysteine ​​as an amino acid does not affect cell culture.

[0176] Similar results were obtained when L-cysteine ​​was added in a double amount (0.14%).

[0177] The present invention provides a basal medium for animal cells that is less expensive and has a lower environmental impact. By utilizing the medium and the method for culturing animal cells of the present invention, it is now possible to provide processed foods containing animal cells at a lower cost and with a lower environmental impact.

Claims

1. A composition for a basal medium for animal cells, comprising a yeast extract, amino acids, inorganic salts and sugars, wherein the amino acids in the composition comprise cystine and / or cysteine, and glutamine.

2. A basal medium composition for animal cells according to claim 1, wherein the total amino acid content in the composition is 0.002 to 1.0 parts by mass when the basal medium composition is taken as 100.

3. A composition for a basal medium for animal cells according to claim 1 or 2, wherein the ratio of cystine and / or cysteine, and glutamine to the total amino acids in the composition is 50% or more.

4. A basal medium composition for animal cells according to any one of claims 1 to 3, wherein the content of cystine and / or cysteine, and glutamine in the composition is 0.001 to 1.0 parts by mass when the basal medium composition is taken as 100.

5. A composition for a basal medium for animal cells according to any one of claims 1 to 4, wherein the content of cystine and / or cysteine, and glutamine in the composition is 0.1 to 5.0 parts by mass per solid content of the composition for a basal medium.

6. A basal medium composition for animal cells according to any one of claims 1 to 5, wherein the content of cystine and / or cysteine ​​in the composition is 0.0001 to 0.02 parts by mass when the basal medium composition is taken as 100.

7. A basal medium composition for animal cells according to any one of claims 1 to 6, wherein the content of cystine and / or cysteine ​​in the composition is 0.005 to 1.0 parts by mass per solid content of the basal medium composition.

8. A basal medium composition for animal cells described in any one of claims 1 to 7, wherein the content of glutamine in the composition is 0.001 to 1.0 parts by mass when the basal medium composition is taken as 100.

9. A basal medium composition for animal cells 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 mass per solid content of the basal medium composition.

10. A composition for a basal medium for animal cells according to any one of claims 1 to 9, wherein the yeast extract is a high amino acid type yeast extract.

11. A basal medium composition for animal cells according to any one of claims 1 to 10, wherein the total amino acid content of the yeast extract is 0.75 parts by mass or more when the basal medium composition is taken as 100 parts by mass.

12. A composition for a basal medium for animal cells according to any one of claims 1 to 11, wherein the yeast extract is a low-nucleic acid type yeast extract.

13. A basal medium composition for animal cells described in any one of claims 1 to 12, wherein the total nucleic acid content of the yeast extract is 2.0 parts by mass or less when the basal medium composition is taken as 100 parts.

14. A composition for a basal medium for animal cells according to any one of claims 1 to 13, wherein the yeast extract is a yeast extract of high amino acid and low nucleic acid content.

15. A basal medium composition for animal cells according to any one of claims 1 to 14, wherein the content of the yeast extract is 0.05 to 20.0 parts by mass when the basal medium composition is taken as 100 parts by mass.

16. A composition for a basal medium for animal cells according to claim 1 or 2, further comprising a vitamin.

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

18. The culture method according to claim 17, wherein the yeast extract is an extract of a high amino acid type yeast, an extract of a low nucleic acid type yeast, or an extract of a high amino acid type and a low nucleic acid type yeast.

19. An animal cell cultured by the culture method according to claim 17 or 18.

20. A processed food comprising the animal cell of claim 19.

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