Animal cell proliferation promoter

Abstract

The purpose of the present invention is to provide a material that can promote the proliferation of animal cells through addition to a serum-reduced culture medium.　It was found that a plant protein material having a specified molecular weight distribution and NSI promoted the proliferation of animal cells through addition at a low concentration.

Description

Animal cell proliferation promoter

[0001] The present invention relates to an animal cell proliferation promoter.

[0002] Efficient cell growth in animal cell cultures will broaden the scope of research and development in various fields. For example, it is expected to be used efficiently in the fields of regenerative medicine and cell-based cultured meat. It may also be possible to improve the productivity of essential substances such as hormones, antibodies, and enzymes for life support. Conventional techniques commonly use serum derived from animals such as cows and horses, or components derived from animal serum, as growth factors in culture media. However, in the fields of regenerative medicine and cell-based cultured meat, which have been gaining attention in recent years, concerns have arisen over the safety of unwanted substances contained in animal serum, the composition of which is not fully understood, and the quality stability of individual animals. Therefore, in recent years, serum-free media and chemically synthesized growth factors have been developed and are being used as alternatives to animal serum and added to culture media.

[0003] The growth factor components used in culture media instead of animal serum are chemically synthesized and expensive. The inedible nature of these growth factors, coupled with their high cost, increases production costs, limiting their use in the development of cultured meat, a potential new food option for the future. Furthermore, while amino acids and low-molecular-weight peptides are added to culture media as nitrogen sources, these components contribute to cell growth as nutrients, and it is difficult for low-molecular-weight components alone to fully replace the functions of serum. Patent Document 1 proposes adding soy protein hydrolysate and yeast extract to serum-free media to increase cell growth rates. Patent Document 2 also discloses technology related to a culture medium for animal cell culture containing a hydrolysate of β-conglycinin concentrate.

[0004] JP-T-2002-520014 A JP-A-2011-182736 A

[0005] An object of the present invention is to provide a material that can promote the growth of animal cells by adding it to a serum-reduced medium.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems, and while examining various protein materials, they have discovered that a protein material with a specific molecular weight distribution and NSI, which differs from conventional soybean proteins, promotes the growth of animal cells even when added at low concentrations, which led to the completion of the present invention.

[0007] That is, the present invention provides: (1) an animal cell proliferation promoter containing a protein material having all of the following characteristics a) to c): a) a protein content in the solid content of 70% by mass or more; b) an NSI of 80 or more; c) a molecular weight distribution measurement result in which the area ratio of 2,000 Da or more and less than 20,000 Da is 30% or more and the area ratio of 20,000 Da or more is 70% or less; (2) the animal cell proliferation promoter according to (1), wherein the protein material has an area ratio of 2,000 Da or more and less than 20,000 Da in a molecular weight distribution measurement result of 45 to 90%; (3) the animal cell proliferation promoter according to (2), wherein the area ratio of less than 2,000 Da in a molecular weight distribution measurement result of 45% or less; (4) a medium for animal cell culture containing the animal cell proliferation promoter according to (1) as a protein material in an amount of 0.015% by weight or more in the medium; (5) A medium for animal cell culture containing the animal cell growth promoter according to (2) as a protein material in an amount of 0.015% by weight or more in the medium; (6) A medium containing the animal cell growth promoter according to (3) as a protein material in an amount of 0.(7) The animal cell culture medium according to (4), wherein the animal cell culture medium is a serum-reduced medium having a serum content of less than 10%, (8) The animal cell culture medium according to (5), wherein the animal cell culture medium is a serum-reduced medium having a serum content of less than 10%, (9) The animal cell culture medium according to (6), wherein the animal cell culture medium is a serum-reduced medium having a serum content of less than 10%, (10) The animal cell culture medium according to (7), wherein the serum-reduced medium is a serum-free medium, (11) The animal cell culture medium according to (8), wherein the serum-reduced medium is a serum-free medium, (12) The animal cell culture medium according to (9), wherein the serum-reduced medium is a serum-free medium, (13) A method for culturing animal cells, comprising culturing animal cells in the animal cell culture medium according to (4). (14) A method for culturing animal cells in the animal cell culture medium according to (5). (15) A method for culturing animal cells in the animal cell culture medium according to (6). (16) A method for culturing animal cells in the animal cell culture medium according to (7). (17) A method for culturing animal cells in the animal cell culture medium according to (10). (18) A method for promoting animal cell growth by adding the animal cell growth promoter according to (1) to an animal cell culture medium and culturing the animal cells. (19) A method for promoting animal cell growth by adding the animal cell growth promoter according to (1) to a serum-reduced animal cell culture medium having a serum content of less than 10% and culturing the animal cells. (20) A method for promoting animal cell growth according to (19), wherein the serum-reduced animal cell culture medium is a serum-free animal cell culture medium.

[0008] According to the present invention, it is possible to promote the growth of animal cells in a serum-reduced medium.

[0009] 1 is a graph showing the results of a cell proliferation test using the mouse striated muscle-derived myoblast cell line C2C12 with plant protein material A. The vertical axis of the graph represents the cell proliferation capacity (fold) of the test group, with the proliferation capacity of the mouse striated muscle-derived myoblast cell line C2C12 cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS (fetal bovine serum), used as the basal medium, set to 1. This graph also shows the results of a cell proliferation test using the mouse striated muscle-derived myoblast cell line C2C12 with plant protein material B. The vertical axis of the graph represents the cell proliferation capacity (fold) of the test group, with the proliferation capacity of the mouse striated muscle-derived myoblast cell line C2C12 cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS (fetal bovine serum), used as the basal medium, set to 1. 1 is a graph showing the results of a cell proliferation test using the mouse striated muscle-derived myoblast cell line C2C12 and vegetable protein material A or an isolated soy protein hydrolysate. The vertical axis of the graph represents the cell proliferation potential (fold) of the test group, with the proliferation potential of the mouse striated muscle-derived myoblast cell line C2C12 cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS (fetal bovine serum), used as the basal medium, set to 1. This graph shows the results of a cell proliferation test using the human proximal tubule cell line HK2 and vegetable protein material A. The vertical axis of the graph represents the cell proliferation potential (fold) of the test group, with the proliferation potential of the human proximal tubule cell line HK2 freshly cultured in the BIO-MPM-1 serum-free medium, used as the basal medium, set to 1.

[0010] (Animal cell proliferation promoter) The animal cell proliferation promoter of the present invention is characterized by containing a vegetable protein material having all of the following characteristics a) to c): a) a protein content in the solid content of 70% by mass or more, b) an NSI of 80 or more, and c) a molecular weight distribution measurement result in an area ratio of 2,000 or more but less than 20,000 of 30% or more and an area ratio of 20,000 or more of 70% or less. Of the solid content of the animal cell proliferation promoter of the present invention, the vegetable protein material preferably accounts for 50% by mass or more, and more preferably 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 100% by mass.

[0011] (Protein Material) The protein material of the present invention can be a vegetable protein material or an animal protein material, with a vegetable protein material being preferred. The concept of the protein material of the present invention is a food material whose main component is vegetable or animal protein and which is used as an ingredient in various processed foods and beverages. Examples of sources of the vegetable protein material include beans such as soybeans, peas, mung beans, lupin beans, chickpeas, kidney beans, lentil beans, and cowpeas; seeds such as sesame seeds, canola seeds, coconut seeds, and almond seeds; grains such as corn, buckwheat, wheat, and rice; vegetables; and fruits. In a more specific embodiment, the vegetable protein material is prepared from bean protein. In an even more specific embodiment, the vegetable protein material is prepared from soybean protein, pea protein, mung bean protein, or broad bean protein. In an even more specific embodiment, the vegetable protein material is prepared from soybean protein or pea protein. As an example, soybean-derived protein materials are prepared by further concentrating and processing proteins from soybean raw materials such as defatted soybeans and whole soybeans, and generally include isolated soybean protein, concentrated soybean protein, powdered soy milk, and various processed versions of these. Examples of animal protein materials include milk proteins such as casein, sodium caseinate, and whey protein, as well as egg white. Vegetable protein materials and animal protein materials can be used in combination. When used in combination, the blending ratio is not particularly limited, but is preferably between 99:1 and 1:99 (vegetable protein material:animal protein material).

[0012] The protein material of the present invention will be further explained using a vegetable protein material as an example.

[0013] a) Protein Purity: The vegetable protein material used in the animal cell culture medium of this embodiment has a protein content of 70% by mass or more, for example, 80% by mass or more, 85% by mass or more, or 90% by mass or more, based on the solid content. As a raw material for the vegetable protein material falling within the above range, isolated proteins are preferred, and when prepared from a protein material derived from soybeans, isolated soybean protein is an example.

[0014] <Measurement of protein purity> Protein purity is measured by the Kjeldahl method. Specifically, the mass of nitrogen measured by the Kjeldahl method is expressed as the protein content in the dry matter in "mass %" relative to the mass of protein material dried at 105°C for 12 hours. The nitrogen conversion factor is 6.25. Basically, it is calculated by rounding off the number to the second decimal place.

[0015] b) Protein NSI The plant protein material used in the animal cell culture medium of this embodiment has an NSI (Nitrogen Solubility Index), used as an index of protein solubility, of 80 or higher. More preferably, an NSI of 85 or higher, 90 or higher, 95 or higher, or 97 or higher can be used. For example, a plant protein material with a high NSI is preferably one that has not undergone any treatment to insolubilize the protein, such as enzymatic hydrolysis or mineral addition, or one that has undergone only minimal treatment, if any. A high NSI of a plant protein material indicates high dispersibility in water and can contribute to the dispersion stability of the animal cell culture medium of this embodiment. An NSI that is too low is undesirable because it tends to cause precipitation. The NSI is expressed as the ratio (mass%) of water-soluble nitrogen (crude protein) to the total nitrogen content, based on the method described below. In the present invention, the NSI is a value measured in accordance with the method described below.

[0016] <Method for Measuring NSI> 60 ml of water is added to 3 g of sample, and the mixture is stirred with a propeller at 37°C for 1 hour. After that, the mixture is centrifuged at 1,400 x g for 10 minutes, and the supernatant (I) is collected. Next, 100 ml of water is added to the remaining precipitate, and the mixture is stirred with a propeller at 37°C for another hour. After that, the mixture is centrifuged and the supernatant (II) is collected. Solutions (I) and (II) are combined, and water is added to the resulting mixture to make 250 ml. This mixture is filtered through filter paper (No. 5), and the nitrogen content (water-soluble nitrogen) of the filtrate is measured by the Kjeldahl method. At the same time, the total nitrogen content in the sample is measured by the Kjeldahl method. The ratio of water-soluble nitrogen to the total nitrogen, expressed as a percentage by mass, is the NSI. The value is generally calculated by rounding to two decimal places.

[0017] c) Molecular Weight Distribution When the molecular weight of the plant protein material used in the animal cell culture medium of this embodiment is measured by gel filtration, the area ratio of the molecular weight distribution is 2,000 Da or more but less than 20,000 Da is 30% or more and 20,000 Da or more but 70% or less. In a specific embodiment, the area ratio of 2,000 Da or more but less than 20,000 Da is 35% or more and 20,000 Da or more but 65% or less. In certain embodiments, when the molecular weight of the plant protein material is measured by gel filtration, the area ratio of the molecular weight distribution of 2,000 Da or more and less than 10,000 Da is 10 to 40%, e.g., 10 to 35%, 15 to 35%, 10 to 30%, or 20 to 30%, and 50 to 80%, e.g., 55 to 75%, 60 to 75%, 60 to 70%, or 65 to 75%, of 10,000 Da or more. In more specific embodiments, the area ratio of less than 2,000 Da is 15% or less, e.g., 5% or less, 13% or less, 9% or less, 8% or less, or 7% or less, with the lower limit being, for example, 0% or more, 1% or more, 1.5% or more, 2% or more, or 3% or more. In another specific embodiment, when the molecular weight of the plant protein material is measured by gel filtration, the area ratio of the molecular weight distribution of 2,000 Da or more and less than 20,000 Da is 45 to 90%, e.g., 50 to 85%, 55 to 80%, 55 to 75%, or 60 to 70%. In a more specific embodiment, the area ratio of less than 2,000 Da is 45% or less, e.g., 40% or less, 35% or less, or 33% or less, with the lower limit being, for example, 0% or more, 1% or more, 2% or more, 5% or more, 10% or more, or 15% or more. In an even more specific embodiment, the area ratio of 10,000 Da or more is less than 50%, e.g., 5 to 45%, 10 to 40%, or 12 to 35%. In even more specific embodiments, the area ratio of 20,000 Da or greater is less than 55%, for example, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, or 15% or less.

[0018] The molecular weight distribution of the plant protein material falling within this range indicates that it is mainly composed of moderately degraded peptides, while highly degraded low molecular weight peptides are rare. The molecular weight distribution is measured by the method described below.

[0019] <Molecular weight distribution> Molecular weight distribution was measured by HPLC (or high performance liquid chromatography). Specifically, the protein material was adjusted to a concentration of 0.1% by mass in the eluent, and the sample solution was filtered through a 0.2 μm filter. A gel filtration system was constructed by connecting two types of columns in series, and first, known proteins, etc., which serve as molecular weight markers as listed in Table 1, were charged, and a calibration curve was created based on the relationship between molecular weight and retention time. Next, the sample solution was charged, and the content ratio (%) of each molecular weight fraction was calculated from the ratio of the area of ​​a specific molecular weight range (time range) to the area of ​​the absorbance chart for the entire chromatogram (1st column: "TSK gel G3000SW"). XL (SIGMA-ALDRICH), 2nd column: "TSK gel G2000SW XL (Sigma-Aldrich), eluent: 1% SDS + 1.17% NaCl + 50 mM phosphate buffer (pH 7.0), 23°C, flow rate: 0.4 ml / min, detection: UV 220 nm).

[0020] (Table 1)

[0021] ("Decomposition and molecular weight distribution adjustment treatment" of plant protein material) The plant protein material used in the animal cell culture medium of this embodiment can be obtained by applying a combination of a "decomposition treatment" that decomposes proteins and a "molecular weight distribution adjustment treatment" that adjusts the molecular weight distribution of proteins. Examples of the "decomposition treatment" include enzyme treatment, pH adjustment treatment (e.g., acid treatment, alkali treatment), heat treatment, cooling treatment, high pressure treatment, organic solvent treatment, mineral addition treatment, supercritical treatment, ultrasonic treatment, electrolysis treatment, and combinations thereof. Examples of the "molecular weight distribution adjustment treatment" include filtration, gel filtration, chromatography, centrifugation, electrophoresis, dialysis, and combinations thereof. The order and number of times of the "decomposition treatment" and the "molecular weight distribution adjustment treatment" are not particularly limited, and the "decomposition treatment" may be performed before the "molecular weight distribution adjustment treatment", or the "molecular weight distribution adjustment treatment" may be performed before the "decomposition treatment", or both treatments may be performed simultaneously. Furthermore, for example, it is possible to perform a "decomposition treatment" between two or more "molecular weight distribution adjustment treatments," to perform a "molecular weight distribution adjustment treatment" between two or more "decomposition treatments," or to perform each treatment multiple times in any order. Note that if the desired molecular weight distribution can be obtained by the "decomposition treatment," the "molecular weight distribution adjustment treatment" does not need to be performed. When these treatments are combined and performed multiple times, all treatments from the raw material may be performed continuously or at intervals. For example, a commercially available product that has undergone a certain treatment may be used as a raw material and subjected to another treatment. Note that, as long as the above-mentioned properties are satisfied, a specific vegetable protein material may be prepared by mixing a vegetable protein material that has undergone a molecular weight distribution adjustment treatment with a protein that has not undergone a molecular weight distribution adjustment treatment. In this case, the ratio of the two (treated protein material:untreated protein) can be adjusted as appropriate within a range that satisfies the above-mentioned properties, and examples of such ratios include a mass ratio of 1:99 to 99:1, 50:50 to 95:5, or 75:25 to 90:10. In one embodiment, the plant protein material used in the animal cell culture medium of this embodiment is a plant protein material that has been subjected to a "degradation and molecular weight distribution adjustment treatment."

[0022] The conditions for the treatment of decomposing or denaturing proteins, such as the type and concentration of enzymes, pH, organic solvent, minerals, etc., temperature, pressure, output intensity, current, and time, can be appropriately determined by those skilled in the art. In the case of enzymes, examples of enzymes that can be used include proteases classified as "metalloproteases," "acid proteases," "thiol proteases," and "serine proteases." The reaction can be carried out at a temperature of 20 to 80°C, preferably 40 to 60°C. In the case of pH adjustment treatment, the treatment can be carried out within a pH range with any of the following upper and lower limits: pH 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, and 12, for example, within a pH range of 2 to 12. In the case of acid treatment, a method of adding an acid or a method of performing a fermentation treatment such as lactic acid fermentation can be used. Examples of acids to be added include inorganic acids such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid, lactic acid, citric acid, gluconic acid, phytic acid, sorbic acid, adipic acid, succinic acid, tartaric acid, fumaric acid, malic acid, and ascorbic acid. Acid may also be added using acid-containing foods and beverages such as lemon juice, concentrated fruit juice, fermented milk, yogurt, and brewed vinegar. For alkali treatment, alkalis such as sodium hydroxide and potassium hydroxide may be added. For denaturant treatment, denaturants such as guanidine hydrochloride, urea, arginine, and PEG may be added. For heating or cooling treatment, examples of heating temperatures include a range of 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, and 150°C, for example, 60°C to 150°C. Examples of cooling temperatures include a range with any of the following temperatures as upper and lower limits: −10° C., −15° C., −20° C., −25° C., −30° C., −35° C., −40° C., −45° C., −50° C., −55° C., −60° C., −65° C., −70° C., and −75° C., for example, −10° C. to −75° C. Examples of heating or cooling times include a range with any of the following times as upper and lower limits: 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 150 minutes, 180 minutes, and 200 minutes, for example, 5 seconds to 200 minutes.In the case of high-pressure treatment, examples of pressure conditions include a range of pressures, with upper and lower limits being any of 100 MPa, 200 MPa, 300 MPa, 400 MPa, 500 MPa, 600 MPa, 700 MPa, 800 MPa, 900 MPa, and 1,000 MPa, for example, 100 MPa to 1,000 MPa. In the case of organic solvent treatment, examples of solvents used include alcohols and ketones, such as ethanol and acetone. In the case of mineral addition treatment, examples of minerals used include divalent metal ions such as calcium and magnesium. In the case of supercritical treatment, treatment can be performed using carbon dioxide in a supercritical state at a temperature of about 30°C or higher and a pressure of about 7 MPa or higher. In the case of ultrasonic treatment, treatment can be performed by irradiating with a frequency of 100 kHz to 2 MHz at an output of 100 to 1,000 W. In the case of electrolysis treatment, treatment can be performed by applying a voltage of 100 mV to 1,000 mV to an aqueous protein solution. In a specific embodiment, the treatment for decomposing the protein is selected from heat treatment and combinations thereof.

[0023] The conditions for the treatment to adjust the molecular weight distribution of proteins, such as the type of filter medium, gel filtration carrier, centrifugation rotation speed, current, time, etc., can be appropriately determined by those skilled in the art. Examples of filter medium include filter paper, filter cloth, diatomaceous earth, ceramic, glass, membrane, etc. Examples of carriers for gel filtration include dextran, agarose, etc. Examples of centrifugation conditions include 1,000 to 3,000 × g, 5 to 20 minutes, etc.

[0024] In addition, conventional commercially available soy protein ingredients such as "Fujipro E," "Fujipro CL," "Fujipro AL," "New Fujipro 4500," "Prolina RD-1," "Prolina 900," and "Prolina HD101R" do not qualify as vegetable protein ingredients that satisfy all of the above characteristics a) to c).

[0025] (Animal Cell Culture Medium) The animal cell culture medium of the present invention comprises the animal cell growth promoter. The animal cell culture medium of the present invention is preferably a serum-reduced medium. One embodiment of the serum-reduced medium can be a serum-reduced medium. In the present invention, a serum-reduced medium is one in which the serum content is reduced compared to a normal serum medium, preferably having a serum content of less than 10%, more preferably 9.5%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. Another embodiment of the serum-reduced medium can be a serum-free medium.

[0026] The type of culture medium is not particularly limited, and commercially available media can be used. The preferred medium varies depending on the type of animal cells to be cultured, and can be appropriately selected by those skilled in the art. Examples of animal cells include fibroblasts, epidermal cells, mammary gland cells, adipocytes, myoblasts, smooth muscle cells, endothelial cells, osteoblasts, hepatocytes, vascular endothelial cells, and their precursor cells. Other examples include somatic stem cells such as hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, as well as pluripotent stem cells and induced pluripotent stem cells. Other examples include animal cells that produce useful substances such as antibodies, enzymes (e.g., urokinase), hormones (e.g., insulin), cytokines (e.g., interferons, interleukins, tumor necrosis factors, colony-stimulating factors, growth factors), and other physiologically active proteins and peptides. Examples of such animal cells include non-transformed cells such as skin cells, chondrocytes, hepatocytes, pancreatic cells, and kidney cells, as well as transformed cells into which genes encoding or genes involved in the biosynthesis of useful substances have been introduced. Examples of transformed cells include antibody-producing cells such as mouse myeloma cells and Chinese hamster ovary cells. These animal cells can be cultured to efficiently produce useful substances. Furthermore, skin cells, cartilage cells, liver cells, pancreatic cells, ES cells, iPS cells, and the like can be cultured and efficiently proliferated, allowing them to be used in the field of regenerative medicine. The animal cells are preferably derived from mammals, such as mice, rats, rabbits, humans, horses, cows, monkeys, pigs, chickens, ducks, dogs, sheep, cats, and goats.

[0027] The amount of the animal cell growth promoter of the present invention in a medium varies depending on the type of animal cells to be cultured, but is desirably 0.015% by mass or more of the protein material in the medium for animal cell culture. Preferably, it is 0.02% by mass or more, or 0.04% by mass or more. The upper limit is preferably 0.5% by mass or less, more preferably 0.4% by mass or less, and even more preferably 0.3% by mass or less, 0.2% by mass or less, 0.15% by mass or less, or 0.1% by mass or less. Specific examples include 0.015 to 0.5% by mass, 0.015 to 0.4% by mass, 0.015 to 0.3% by mass, 0.015 to 0.2% by mass, 0.015 to 0.15% by mass, 0.015 to 0.1% by mass, 0.02 to 0.5% by mass, 0.02 to 0.4% by mass, 0.02 to 0.3% by mass, 0.02 to 0.2% by mass, 0.02 to 0.15% by mass, 0.02 to 0.1% by mass, 0.04 to 0.5% by mass, 0.04 to 0.4% by mass, 0.04 to 0.3% by mass, 0.04 to 0.2% by mass, 0.04 to 0.15% by mass, and 0.04 to 0.1% by mass. By containing the protein material of the present invention in an amount within the above range, proliferation of animal cell cultures can be promoted. In particular, the protein material of the present invention is advantageous in that it can promote the proliferation of animal cells even at low concentrations of 0.1% by mass or less, as described above. In the present invention, promoting the proliferation of animal cells means that the proliferation of cells cultured in a medium supplemented with the protein material of the present invention is higher than the proliferation of cells cultured in a medium without the protein material. Specifically, when the proliferation ability of cells cultured in a medium without the protein material is set to 1, the proliferation ability of cells cultured in a medium supplemented with a plant protein material is 1.07 or higher. Preferably, the proliferation ability is 1.08 or higher, more preferably 1.10 or higher, even more preferably 1.13 or higher, and even more preferably 1.15 or higher. The method for measuring cell proliferation will be described below.

[0028] (Growth-promoting effect on animal cells) When animal cells are cultured in the animal cell culture medium of the present invention, the protein material of the present invention promotes cell growth of the animal cells. As a result, if the animal cells whose growth is promoted are capable of producing a substance useful for the production of cultured meat, the production of cultured meat by the animal cells can also be promoted. Furthermore, if the animal cells whose growth is promoted are of a type that produces a substance useful to the human body, the production of the useful substance by the animal cells can also be promoted. Various useful substances produced by animal cells include antibodies, as well as enzymes and hormones.

[0029] The present invention will be described below by way of examples, in which parts and percentages are by weight unless otherwise specified.

[0030] (Preparation of vegetable protein materials) The following vegetable protein materials were obtained and prepared. The protein content of all of these vegetable protein materials was 80% by mass or more. Detailed analytical values ​​are shown in Table 2. Vegetable protein material A: isolated soy protein decomposed and treated with molecular weight distribution adjustment (test product manufactured by Fuji Oil Co., Ltd., raw isolated soy protein: FujiPro F (commercially available product by Fuji Oil Co., Ltd.)) Vegetable protein material B: isolated soy protein decomposed and treated with molecular weight distribution adjustment (test product manufactured by Fuji Oil Co., Ltd., raw isolated soy protein: FujiPro F (commercially available product by Fuji Oil Co., Ltd.)) Isolated soy protein hydrolysate: commercially available product by Fuji Oil Co., Ltd.

[0031] (Table 2)

[0032] (Example 1) Proliferation promoting effect on mouse striated muscle-derived myoblasts The proliferation promoting effect of plant protein material A on mouse striated muscle-derived myoblasts was confirmed by the following method.

[0033] (Cell culture method) Dulbecco's modified Eagle's medium (hereinafter referred to as DMEM) containing 5% FBS (fetal bovine serum) and penicillin-streptomycin-amphotericin B suspension was prepared at a 100-fold dilution. Mouse striated muscle-derived myoblast cell line C2C12 (hereinafter referred to as C2C12) was cultured in this medium at a concentration of 5 × 10 4A cell suspension adjusted to 0.1 cells / mL was obtained. The resulting cell suspension was added to 8 wells of a 96-well plate at 0.1 mL / well. After equilibration at 37°C for 24 hours (CO2 concentration = 5%), the medium was replaced with DMEM containing 5% FBS supplemented with various concentrations of plant protein material A. This medium was then cultured at 37°C for 48 to 72 hours (CO2 concentration = 5%), after which cell proliferation was measured. Cultures were also performed in DMEM containing 5% FBS or 10% FBS without plant protein material. Plant protein material A was added at 0.2 g / L (equivalent to 0.02%) to 1.5 g / L (equivalent to 0.15%) to DMEM containing 5% FBS.

[0034] (Method for assessing cell proliferation) Cell proliferation was measured using Cell Counting Kit-8 manufactured by Dojindo Laboratories, Inc. Specifically, water-soluble tetrazolium salt (WST-8) was added to each culture medium after culturing, and after incubation at 37°C for 60 minutes, absorbance was measured at 450 nm. The measurement principle is that NADH produced by dehydratase in the cells reduces the water-soluble tetrazolium salt (WST-8) to orange-colored water-soluble formazan via an electron carrier. The absorbance of this formazan at 450 nm is proportional to the number of viable cells, allowing the number of viable cells to be measured.

[0035] The results are shown in Figure 1. Compared to cell proliferation in DMEM containing standard 10% FBS, proliferation in DMEM containing 5% FBS, which has half the FBS concentration, was clearly reduced. However, the reduced proliferation was significantly restored by adding 0.2 g / L (equivalent to 0.02%) to 1.5 g / L (equivalent to 0.15%) of vegetable protein material A to the 5% FBS-containing DMEM. This confirms that vegetable protein material A has a proliferation effect on C2C12 in serum-reduced medium.

[0036] Example 2: Cells were cultured and their proliferation ability was evaluated in the same manner as in Example 1, except that plant protein material B was used as the plant protein material and added at 1.0 g / L to DMEM containing 5% FBS. The results are shown in Figure 2. It was confirmed that plant protein material B also had a proliferation effect on C2C12 in serum-reduced medium.

[0037] (Example 3, Comparative Example 1) Cells were cultured in the same manner as in Example 1, except that plant protein material A or isolated soy protein hydrolysate was used as the plant protein material and added at 0.5 g / L to DMEM containing 5% FBS. The proliferation ability was evaluated. The results are shown in Figure 3. Plant protein material A was confirmed to have a high proliferation effect on C2C12 in serum-reduced medium (Example 3, labeled "protein material A" in the figure). However, an isolated soy protein hydrolysate whose molecular weight distribution did not satisfy the numerical value specified in the present invention had a low proliferation effect on C2C12 (Comparative Example 1, labeled "isolated soy protein hydrolysate" in the figure), and the results did not satisfy the acceptance criteria of the present invention.

[0038] (Example 4, Comparative Example 2) The effect of the plant protein material of the present invention in a serum-free medium was confirmed by the following procedure.

[0039] A serum-free medium (BIO-MPM-1, manufactured by Biological Industries Ltd.) containing 5 μM / mL fibronectin (an adhesion factor) and supplemented with glutamine to a concentration of 2 mM was prepared. 1 × 10 human proximal tubule cell line HK2 was cultured in this medium. 4 A cell suspension adjusted to 0.125 g / L (equivalent to 0.0125%) to 5 g / L (equivalent to 0.5%) of plant protein material A was obtained. The resulting cell suspension was added to 8 wells of a 96-well plate at 0.1 mL / well. After equilibration at 37°C for 24 hours (CO2 concentration = 5%), the medium was replaced with serum-free medium (BIO-MPM-1) supplemented with various concentrations of plant protein material A. After culturing at 37°C for 48 to 72 hours (CO2 concentration = 5%), cell proliferation was measured. Plant protein material A was added to the BIO-MPM-1 serum-free medium at concentrations ranging from 0.125 g / L (equivalent to 0.0125%) to 5 g / L (equivalent to 0.5%). Cell proliferation was evaluated in the same manner as in Example 1. The evaluation results are shown in Figure 4. It was confirmed that when the plant protein material A was added in amounts of 0.5 g / L, 1.25 g / L, 2.5 g / L, and 5 g / L (Example 4), the proliferation effect of the human proximal tubule cell line HK2 in serum-free medium was high.

Claims

1. An animal cell proliferation promoter containing a protein material having all of the characteristics described in a) to c) below. a) Protein content in solids is 70% by mass or more, b) NSI is 80 or higher, c) In the measurement results of the molecular weight distribution, the area ratio of 2,000 Da or more and less than 20,000 Da is 30% or more, and the area ratio of 20,000 Da or more is 70% or less.

2. The animal cell proliferation promoter according to claim 1, wherein the protein material has an area ratio of 45-90% of molecules with a molecular weight of 2,000 Da or more and less than 20,000 Da, as determined by molecular weight distribution measurement.

3. The animal cell proliferation promoter according to claim 2, wherein the area ratio of less than 2,000 Da, as measured by molecular weight distribution, is 45% or less.

4. A culture medium for animal cells containing 0.015% by weight or more of the animal cell proliferation promoter described in any one of claims 1 to 3 as a protein material.

5. The animal cell culture medium according to claim 4, wherein the animal cell culture medium is a culture medium in which the amount of serum in the medium is reduced to less than 10%.

6. The animal cell culture medium according to claim 5, wherein the culture medium with reduced serum content is a serum-free medium.

7. A method for culturing animal cells, comprising culturing animal cells in the animal cell culture medium described in claim 4.

8. A method for culturing animal cells, comprising culturing animal cells in the animal cell culture medium described in claim 5.

9. A method for culturing animal cells, comprising culturing animal cells in the animal cell culture medium described in claim 6.

10. A method for promoting the proliferation of animal cells, comprising adding the animal cell proliferation promoter described in claim 1 to an animal cell culture medium and culturing the animal cells.

11. A method for promoting the proliferation of animal cells, comprising adding the animal cell proliferation promoter described in claim 1 to an animal cell culture medium in which the serum content has been reduced to less than 10%, and culturing the animal cells.

12. A method for promoting the proliferation of animal cells according to claim 10, wherein the animal cell culture medium with reduced serum is a serum-free animal cell culture medium.