Medium for cell culture, antibacterial agent for medium for cell culture, and antibacterial method for medium for cell culture

Abstract

The present invention provides technology relating to a medium for cell culture which is excellent in antibacterial properties without using an antibiotic. One embodiment of the present invention is a medium for cell culture. The medium for cell culture includes yolk or a purified yolk product, glycine, and a basal medium.

Description

Cell culture medium, antibacterial agent for cell culture medium, and antibacterial method for cell culture medium

[0001] The present invention relates to a cell culture medium, an antibacterial agent for a cell culture medium, and an antibacterial method for a cell culture medium.

[0002] Conventionally, known cell culture media include those in which various additives are added to a basal medium containing amino acids, vitamins, etc. For example, Patent Document 1 discloses a medium for animal cell culture in which an egg yolk-derived extract and a specific organ extract are added to a predetermined basal medium.

[0003] Japanese Patent Application Publication No. 5-308962

[0004] Conventional cell culture media themselves do not have sufficient antibacterial properties, and in order to impart antibacterial properties to cell culture media, it is necessary to separately add antibiotics such as penicillin, ampicillin, and streptomycin. However, these antibiotics may not be suitable for use depending on the cells being cultured, such as in cultured meat, and are therefore not suitable for widespread use in cell culture. The present invention has been developed in consideration of the above-mentioned problems and aims to provide antibacterial properties without the use of antibiotics.

[0005] One aspect of the present invention is a cell culture medium. The cell culture medium comprises 0.25% (mass / mass percent) or more of glycine and 5.0 to 80.0% (mass / mass percent) of egg yolk or purified egg yolk. The cell culture medium of the above aspect may also comprise 0.25 to 1.0% (mass / mass percent) of glycine and 5.0 to 50.0% (mass / mass percent) of egg yolk or purified egg yolk. The cell culture medium of the above aspect may be used for culturing mammalian, avian, or fish cells. The cell culture medium of the above aspect may also be used for culturing cells for cultured meat. Another aspect of the present invention is an antibacterial agent for cell culture medium. The antibacterial agent for cell culture medium comprises egg yolk or purified egg yolk and glycine as active ingredients. Yet another aspect of the present invention is an antibacterial method for cell culture medium. The antibacterial method for the cell culture medium includes the step of blending egg yolk or a purified egg yolk product and glycine into a basal medium.

[0006] According to the present invention, a technology can be provided relating to a cell culture medium that has excellent antibacterial properties without using antibiotics.

[0007] Figure 1 is a micrograph showing bacterial growth in the media of Comparative Example 15, Examples 14, and 32. Figure 2 is a photograph showing colony formation in a medium containing egg yolk (Example 14) and a medium not containing egg yolk (Comparative Example 2). Figure 3 is a graph showing the time course of antibacterial activity in the media of Examples 14, Comparative Examples 7, and 16. Figure 4 is a micrograph showing cell growth in the media of Example 14 and Comparative Example 7.

[0008] Hereinafter, embodiments of the present invention will be described in detail. In this specification, the expression "a to b" in the description of a range of values ​​means that the range is from a to b, unless otherwise specified.

[0009] (Cell Culture Medium) A cell culture medium according to an embodiment contains glycine and egg yolk or purified egg yolk.

[0010] The content of glycine in the cell culture medium is preferably 0.25% (mass / mass percent) or more, and more preferably 0.25 to 1.0% (mass / mass percent).

[0011] The content of egg yolk or purified egg yolk in the cell culture medium is preferably 5.0 to 80.0% (mass / mass percent), more preferably 5.0 to 50.0% (mass / mass percent). This refers to egg yolk contained in eggs of birds, fish, amphibians, etc., specifically, the yolk contained in eggs of chickens, ducks, quails, etc. These may be used alone or in combination of two or more. Of these, chicken yolk is preferably used as the egg yolk from the viewpoints of low cost and easy availability. The purified egg yolk is a purified product obtained by extracting components such as a phospholipid-protein complex, vitamins, amino acids, inorganic salts, fatty acids, and dietary fiber, which are part of the yolk.

[0012] By adjusting the glycine content in the cell culture medium to 0.25% (mass / mass percent) or more and the egg yolk or purified egg yolk content in the cell culture medium to 5.0 to 80.0% (mass / mass percent), antibacterial properties can be exhibited without the use of antibiotics. Here, antibacterial properties refer to the property of inhibiting the growth or reproduction of bacteria without killing them.

[0013] Furthermore, by setting the content of glycine in the cell culture medium to 0.25 to 1.0% (mass / mass percent) or more and the content of egg yolk or purified egg yolk in the cell culture medium to 5.0 to 50.0% (mass / mass percent), it is possible to improve cell growth while exhibiting antibacterial properties without using antibiotics.

[0014] The cell culture medium according to the embodiment may contain a basal medium in addition to the above-mentioned glycine and egg yolk. The basal medium contains specific amino acids and vitamins. Examples of amino acids include asparagine, aspartic acid, cystine, glutamic acid, hydroproline, and proline. Examples of vitamins include folic acid, inositol, nicotinamide, pyridoxylated hydrochloride, riboflavin, and thiamine hydrochloride. Specific examples of basal media will be described later.

[0015] The cell culture medium according to the embodiment may also contain other components known as components of cell culture media in appropriate amounts as long as the effects are not impaired. Examples of other components include inorganic salts, nitrogen sources, enzyme inducers, and the like. Examples of inorganic salts include phosphates, magnesium salts, potassium salts, sodium salts, iron salts, cobalt salts, and other trace metal salts. Examples of phosphates include diammonium hydrogen phosphate and dipotassium hydrogen phosphate. Examples of magnesium salts include magnesium sulfate. Examples of nitrogen sources include ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and sodium glutamate. Examples of enzyme inducers include urea, methylurea, 1,1-dimethylurea, 1,3-dimethylurea, ethylurea, 1,1-diethylurea, 1,3-diethylurea, and methacrylamide.

[0016] The solvent used in the cell culture medium according to the embodiment is not particularly limited, and may be, for example, purified water, distilled water, ion-exchanged water, etc. The cell culture medium according to the embodiment may be in a gel form.

[0017] The cell culture medium according to the embodiment may be bacteriostatic, particularly against Gram-positive bacteria, such as Bacillus cereus, Bacillus anthracis, Bacillus subtilis, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Clostridium tetani, Clostridium butyricum, Clostridium difficile, Lactococcus lactis, Corynebacterium diphtheriae, Listeria monocytogenes, and Streptococcus pneumoniae.

[0018] Furthermore, the cell culture medium according to the embodiment may contain cell growth factors or cell growth factors such as IGF, FGF, HGF, VGEF, EGF, NGF, TGF, BDNF, PDGF, etc., within the range that does not inhibit the effect, thereby further promoting the proliferation or growth of cells cultured in the cell culture medium.

[0019] The pH of the cell culture medium according to the embodiment is not particularly limited, but is preferably 4 to 10, more preferably 6 to 8.5.

[0020] (Uses) The cell culture medium according to the embodiment is not limited to a particular cell type and can be used to culture a variety of cells, such as mammalian, avian, or fish cells. In particular, the cell culture medium according to the embodiment exhibits antibacterial properties without the need for antibiotics, making it suitable for use in culturing cells for cultured meat.

[0021] (Antibacterial Agent for Cell Culture Medium) The antibacterial agent for cell culture medium according to the embodiment contains egg yolk or a purified egg yolk product, and glycine. In addition to the above-described active ingredients, the antibacterial agent for cell culture medium may also contain preservatives, shelf-life extenders, antiseptics, disinfectants, antioxidants, pH adjusters, emulsifiers, etc. When the antibacterial agent for cell culture medium according to the present embodiment is blended into a basal medium, the amounts of the antibacterial agent for cell culture medium are adjusted so that the egg yolk or purified egg yolk product and glycine blended into the basal medium are in the above-described blend amounts, thereby imparting antibacterial properties to the basal medium without the use of antibiotics.

[0022] (Antibacterial method for cell culture medium) The antibacterial method for cell culture medium according to the embodiment includes a step of blending egg yolk or a purified egg yolk product and glycine into a basal medium. The egg yolk or purified egg yolk product and glycine blended into the basal medium are as described above. This allows the basal medium to have antibacterial properties without using antibiotics.

[0023] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations can also be adopted.

[0024] EXAMPLES The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited to these.

[0025] (Effective glycine concentration and concentration of egg yolk or purified egg yolk product) <Conditions for implementation> Glycine and egg yolk were mixed in the amounts shown in Table 1, and 100 μL of the basal medium below was added to a 96-well plate to prepare a cell culture medium. Glycine, a food additive manufactured by Nippon Garlic Co., Ltd., was used. In Examples 1-31, glycine was mixed to a concentration of 0.25 to 2.00% (w / w). B. cereus was collected by Integriculture. For I-MEM, a basal medium sold by Integriculture was used. For DMEM, a basal medium from Fujifilm Wako Pure Chemical Industries was used.

[0026] <Bacteriostasis Test (A)> Glycine was dissolved in DMEM to a final concentration of 0.0-2.0% (w / w). The mixture was sterilized by filtration using a PVDF filter (0.22 μm). Purified egg yolk (a chicken egg yolk was removed and the vitelline membrane was removed. An equal volume of 1% sodium chloride solution was then added and mixed. This solution was centrifuged at 20,000 × g for 30 minutes at 4°C, and the resulting supernatant was added to a concentration equivalent to egg yolk of 0-50% (w / w). This was used as the prepared medium. A B. cereus suspension (isolated from a microbially contaminated cell culture system and suspended in Integriculture's cell culture medium (hereinafter referred to as I-MEM) containing 10% (w / w) purified egg yolk) was diluted with the prepared medium to a final concentration of 2.0 × 10 ４ 100 μL of the solution was added to a 96-well plate and incubated at 37°C, CO ２ The cells were cultured at 5% CO₂ for 4 days. ＴＭ 100 μL of Microbial Cell Viability Assay (Promega) reagent was added, and the mixture was incubated for 30 minutes at 37°C in the dark. Luminescence (RLU) was measured using a Plate Reader (PerkinElmer, Nivo Microplate Reader), and the antibacterial activity was evaluated based on the growth of the bacteria. The relative value was determined by setting the RLU of 0.00% (w / w) glycine and 0% (w / w) purified egg yolk as 100%, and the turbidity of the medium was classified and described according to the following criteria: Excellent: RLU (relative value) less than 50%; Good: RLU (relative value) 50% or more and 90% or less; Fair: RLU (relative value) more than 90%

[0027] <Cell proliferation (B)> Glycine was dissolved in DMEM to a final concentration of 0.0-2.0% (w / w). The mixture was sterilized by filtration using a PVDF filter (0.22 μm). Purified egg yolk was added to a concentration equivalent to 0.0-99.0% (w / w) of egg yolk. This was used as the prepared medium. Duck liver-derived cell suspension (the cells were subcultured in I-MEM containing 10% (w / w) purified egg yolk, detached from the bottom with trypsin, and suspended in I-MEM) was diluted to 1.0 x 10 cells using the prepared medium. ５ 100 μL of the solution was added to a 96-well plate and incubated at 37°C in CO ２The cells were cultured at 5% CO₂ for 4 days. 100 μL of CellTiter-Glo® 2.0 Cell Viability Assay (Promega) reagent was added, and the reaction was allowed to proceed for 30 minutes at 37°C in the dark. Cell proliferation was assessed by measuring luminescence using a Plate Reader (PerkinElmer, Nivo microplate reader). The cell proliferation was assessed based on the relative RLU of 0% (w / w) glycine and 10% (w / w) purified egg yolk, defined as 100%. The classification is shown below: Excellent: RLU (relative value) > 99% Good: RLU (relative value) ≥ 90% and ≤ 99% Fair: RLU (relative value) < 90% Poor: No cell proliferation. Furthermore, the "bacteriostasis (A) + cell proliferation (B)" in Table 1 was evaluated according to the following criteria. ◎: Bacteriostasis (A) is "good" or better and cell proliferation (B) is "good" or better. 〇: Bacteriostasis (A) is "fair" or cell proliferation (B) is "fair", except when cell proliferation (B) is "poor". ×: Cell proliferation (B) is "poor".

[0028] (Three combinations of glycine, egg yolk or purified egg yolk, and basal medium) Glycine and egg yolk extract were added to each solvent (PBS, I-MEM, DMEM) in the combinations shown in the table. After dissolving glycine, the mixture was sterilized by filtration using a PVDF filter (0.22 μm), and then purified egg yolk was added. A B. cereus suspension (isolated from a cell culture system contaminated with microorganisms and suspended in I-MEM containing 10% (w / w) purified egg yolk) was diluted with 2.0 x 10 B. cereus cells using the prepared medium. ４ 100 μL of the solution was added to a 96-well plate and incubated at 37°C, CO ２ The samples were cultured at 5% CO for 4 days. Before and after the culture, the optical density was measured at 600 nm using a Plate Reader (PerkinElmer, Nivo Microplate Reader), and samples that showed no increase in turbidity were evaluated as having antibacterial activity (+). For 1% (w / w) glycine, 10% (w / w) egg yolk / I-MEM, 100 μL of the sample solution after the antibacterial treatment was plated on SCD agar medium and incubated at 37°C in CO. ２The cells were cultured at 5% CO₂ for 4 days. The presence or absence of colonies was confirmed visually. In Table 2, "antibacterial activity" is indicated by (+) and "no antibacterial activity" by (-). In addition, for "cell proliferation (B) + antibacterial activity (C)" in Table 2, the evaluation was classified as follows: ◎: Cell proliferation (B) was "good" or better and antibacterial activity (C) was (+); ○: Cell proliferation (B) was "fair" and antibacterial activity (C) was (+); ×: Cell proliferation (B) was "poor" and antibacterial activity (C) was (-).

[0029] (Comparison of cell culture medium application for each food additive) Each raw material was dissolved in I-MEM (Integriculture) to the arbitrary final concentration shown in the table. The solution was sterilized by filtration using a PVDF filter (0.22 μm). Egg yolk purified product was added to a concentration equivalent to egg yolk of 10.0% (w / w). This was used as the prepared medium.

[0030] <Test for evaluating bacterial count based on turbidity> A B. cereus suspension (isolated from a cell culture system contaminated with microorganisms and suspended in I-MEM containing 10% (w / w) purified egg yolk) was immersed in a prepared medium at 2.0 × 10 ４ The solution was diluted to CFU / mL. 100 μL was added to a 96-well plate and incubated at 37°C, 5% CO ２ The samples were cultured at 600 nm for 4 days. Before and after the culture, the optical density was measured at 600 nm using a Plate Reader (PerkinElmer, Nivo microplate reader), and samples that showed no increase in turbidity were evaluated as having antibacterial activity (+).

[0031] <Cell culture feasibility study (D)> Duck liver cell suspension (the cells were subcultured in I-MEM containing 10% (w / w) purified egg yolk, detached from the bottom with trypsin (Thermo Fisher Scientific), and suspended in I-MEM) was cultured at 1.0 x 10 ５ 100 μL was added to a 96-well plate and incubated at 37°C, 5% CO ２The cells were cultured for 4 days under a microscope before and after the culture. Cells that showed cell growth (reached confluence) were evaluated as having a cell culturability (D) of (+), and those that did not were evaluated as having a cell culturability (-). In addition, in Table 3, "Antibacterial activity (C) + cell culturability (D)" was evaluated according to the following classification: ◎: Antibacterial activity (C) was (+) and cell culturability (D) was (+); ×: Antibacterial activity (C) was (-) or cell culturability (D) was (-).

[0032] (Bactericidal effect) A B. cereus suspension (isolated from a cell culture system contaminated with microorganisms and suspended in I-MEM containing 10% (w / w) purified egg yolk, approximately 3.0 × 10 ７ CFU / mL) was added to the culture medium. ２ After the test, Example 14 was cultured at 5% CO for 7 days, and the antibacterial activity was evaluated visually (a white layer was formed when growth was observed). After the test, 100 μL of the cultured liquid was inoculated onto an SCD agar medium, and then incubated at 37°C and CO ２ The cells were cultured at 5% CO₂ for 4 days, and visual inspection confirmed that no colonies were formed.

[0033] ((Results)) (Range of Bacteriostasis (A)) From Examples 16-18, 27-28, and 31, it was confirmed that egg yolk or purified egg yolk product containing 1.25% (w / w) or less or 45% (w / w) or more was judged to be "Acceptable." From the above, it was demonstrated that a cell culture medium containing 0.25% (w / w) or more of glycine and 1.25 to 40.0% (w / w) of egg yolk or purified egg yolk product has antibacterial properties.

[0034] (Range of Cell Proliferation (B)) With regard to cell proliferation, Examples 11 and 19 confirmed that cell proliferation was "fair" when the egg yolk or purified egg yolk concentration was 40% (w / w). Furthermore, Example 26 confirmed that cell proliferation was "fair" when the egg yolk or purified egg yolk concentration was 1.25% (w / w). Therefore, it was demonstrated that a cell culture medium containing 0.5 to 1.00% (w / w) or more glycine and 1.25 to 25.0% (w / w) egg yolk or purified egg yolk containing components equivalent to the same concentration of egg yolk is a more desirable cell culture medium. Furthermore, Example 8 confirmed that cell proliferation was "good" only when the glycine concentration was 1.5% (w / w). Additionally, Examples 1 and 2 confirmed that cell proliferation was "good" when the glycine concentration was 2% (w / w), regardless of the egg yolk or purified egg yolk concentration. This indicates that the conditions of 1.5% (w / w) glycine and 25% (w / w) egg yolk or purified egg yolk, and the conditions of 2.0% or more glycine and 5 to 10% (w / w) egg yolk or purified egg yolk containing components equivalent to egg yolk at the same concentration, are desirable cell culture media.

[0035] In Table 1, based on the range of bacteriostasis (A) and the range of cell proliferation (B), the most desirable conditions are indicated by ◎, and the next most desirable conditions are indicated by ○.

[0036] (Three combinations of glycine, egg yolk or purified egg yolk, and basal medium) Table 2 shows that Comparative Examples 8 and 11 and Examples 14 and 32 showed a positive antibacterial (C) score. Furthermore, for "cell growth (B) + antibacterial (C)," only Example 14 was rated as excellent, while the other comparative examples were rated as poor. This indicates that cells do not grow satisfactorily in the presence of PBS and no egg yolk or purified egg yolk, as in Comparative Examples 8 and 11. The difference between Comparative Example 15 and Examples 14 and 32 indicates that PBS does not maintain antibacterial activity. Figure 1 shows a photograph of bacterial observation under a microscope. In this image, bacterial growth was confirmed only when the solvent was PBS, and no bacterial growth was observed in the basal media of DMEM and I-MEM. The results of the presence or absence of bacteria on SCD agar medium, with and without egg yolk, confirmed that colony formation was not observed in the presence of egg yolk (Example 14) (Figure 2). This demonstrates the importance of the combination of glycine, egg yolk or purified egg yolk, and basal medium.

[0037] (Comparison of cell culture medium application for each food additive) In Table 3, Example 14 and Comparative Example 16 were marked with a ◎ for "antibacterial activity (C) + cell culturing activity (D)." No components exhibiting both antibacterial activity and cell proliferation were confirmed under other conditions. Comparative Example 16 is generally used as an antibiotic, but cannot be used in food applications. Therefore, glycine, similar to Example 14, is most suitable for food applications.

[0038] (Confirmation of antibacterial activity duration) The results are shown in Figure 3. In Example 14, as in Comparative Example 16, no increase in turbidity was observed. Furthermore, even when the medium was replaced after three days, the turbidity did not increase, and the condition containing glycine and egg yolk showed the same water level as the condition containing antibiotics. Therefore, it was confirmed that the antibacterial effect lasted for three days, and that the effect was maintained by further replacing the medium.

[0039] (Bactericidal Effect) After the culture, microscopic observation was performed, and it was confirmed that cells had proliferated (reached confluence) ( FIG. 4 ). This demonstrated that Example 14 has a bactericidal effect and is also suitable for cell culture.

[0040]

[0041]

[0042]

[0043] The cell culture medium of the present disclosure has excellent antibacterial and bactericidal properties without the use of antibiotics, and can therefore be widely used for cell culture. CROSS-REFERENCE TO RELATED APPLICATIONS

[0044] This application claims priority based on Japanese Patent Application No. 2024-148489, filed with the Japan Patent Office on August 30, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A cell culture medium comprising egg yolk or purified egg yolk, glycine, and a basal medium.

2. A cell culture medium comprising 0.25% (mass / mass percent) or more of glycine and 1.25 to 40.0% (mass / mass percent) of egg yolk or purified egg yolk.

3. The cell culture medium according to claim 1, comprising: 0.25 to 1.5% (mass / mass percent) of glycine; and 1.25 to 25.0% (mass / mass percent) of egg yolk or purified egg yolk.

4. The cell culture medium according to claim 1, which is used for culturing mammalian, avian, or fish cells.

5. The cell culture medium of claim 1, which is used for culturing cells for cultivated meat.

6. An antibacterial agent for cell culture media, comprising egg yolk or purified egg yolk and glycine.

7. An antibacterial method for a cell culture medium, comprising the step of incorporating egg yolk or purified egg yolk and glycine into a basal medium.

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