Serum-free protein-free medium for culturing mycoplasma

Abstract

Provided is a serum-free protein-free medium for culturing mycoplasma, the medium comprising: (a) a basal medium; (b) a lipid mixture containing cholesterol, palmitic acid and oleic acid; (c) a polyvinyl alcohol-based resin; and (d) cyclodextrin.

Description

Serum-free and protein-free medium for culturing mycoplasmas

[0001] The present invention relates to a serum-free, protein-free medium for culturing mycoplasma.

[0002] Mycoplasma bacteria are pathogenic bacteria for both animals and plants. They primarily cause atypical pneumonia in humans, and in livestock, they cause avian mycoplasmosis (bronchial pneumonia, leg arthritis, etc.) and bovine mycoplasmosis (bovine pneumonia, mycoplasmal mastitis, etc.), causing significant damage. Mycoplasma infections can become chronic through repeated reinfection, requiring prompt treatment. Because mycoplasmas lack a cell wall, they are not susceptible to β-lactam antibiotics, requiring treatment with macrolide or tetracycline antibiotics. Therefore, early detection and diagnosis of mycoplasma are crucial for determining initial treatment strategies.

[0003] Mycoplasma detection and diagnosis are primarily performed through culture growth tests. Live and inactivated mycoplasma vaccines for livestock are also available. Because mycoplasma cannot synthesize lipids on its own, serum must generally be added to the culture medium. However, serum is expensive and suffers from significant quality variations between batches. From an ethical perspective, such as animal protection, it is desirable to avoid the use of serum.

[0004] Culture media containing lipids and serum albumin as a lipid carrier instead of serum have been reported (Patent Document 1, Non-Patent Documents 1 and 2). However, serum albumin is also expensive. Furthermore, the large amount of protein contained in the medium can interfere with subsequent analysis after culture.

[0005] International Publication No. 2021 / 078935

[0006] Burgos, R. et al., Microbiol. Spectr., 2023, 11(3):e0485922Gaspari, E. et al., NPJ Syst. Biol. Appl., 2020, 6(1):33

[0007] The present invention has been made with the objective of providing a medium for culturing mycoplasma that is inexpensive and has consistent quality.

[0008] As a result of extensive research, the present inventors have succeeded in identifying supplementary components that enable mycoplasma to be cultured without the use of serum or serum albumin, thereby completing the present invention.

[0009] That is, the present invention provides a serum-free, protein-free medium for culturing mycoplasma, comprising the following components: (a) a basal medium, (b) a lipid mixture containing cholesterol, palmitic acid, and oleic acid, (c) a polyvinyl alcohol-based resin, and (d) cyclodextrin.

[0010] The content of component (b) in the serum-free, protein-free medium is preferably 5 to 120 mg / L.

[0011] The content of component (c) in the serum-free, protein-free medium is preferably 0.2 to 3.0 w / v %.

[0012] The content of component (d) in the serum-free, protein-free medium is preferably 0.2 to 2.0 w / v %.

[0013] The polyvinyl alcohol resin is preferably polyvinyl alcohol.

[0014] The cyclodextrin is preferably a methylated cyclodextrin.

[0015] The basal medium is preferably SP4 medium.

[0016] The medium of the present invention does not contain serum or serum albumin, and is therefore inexpensive and has excellent consistency of quality, making it useful for testing and drug discovery research.

[0017] Figure 1 is a graph showing the growth rate of M. mycoides in SP4 medium (control) and serum-free, protein-free SP4 medium supplemented with various concentrations of lipid, polyvinyl alcohol (PVA), and methyl-β-cyclodextrin (MβCD). Figure 2 is a graph showing the relationship between M. mycoides growth in serum-free, protein-free SP4 medium and the concentrations of lipid and MβCD. Figure 3 is a graph showing the growth rate of M. mycoides in SP4 medium (control), SP4-NT (control), and SP4-wo medium supplemented with varying concentrations of added lipid components. Figure 4 is a graph showing the growth rate of M. mycoides in SP4 medium (control), SP4-NT (control), and SP4-LPC4 medium supplemented with varying concentrations of added lipid components, with varying lipid concentrations. Figure 5 is a graph showing the growth rate of M. mycoides in serum-free, protein-free SP4 medium supplemented with lipid and either BSA, PVA, or MβCD. Figure 6 is a graph showing the growth rate of M. mycoides in serum-free, protein-free SP4 medium supplemented with various concentrations of lipid, PVA, and MβCD. Figure 7 is a graph showing the growth rate of M. mycoides in serum-free, protein-free SP4 medium, serum-free, protein-free R2 medium, serum-free, protein-free mHF medium, and serum-free, protein-free MBB medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 8 is a graph showing colony formation and morphology of M. mycoides on serum-free, protein-free SP4 medium plates containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 9 is a graph showing the growth rate of M. mycoides frozen and thawed using serum-free, protein-free SP4 medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 10 shows SDS-PAGE images (CBB staining) of SP4-based serum-containing or serum-free, protein-free media. Figure 11 shows graphs showing the growth rates of M. gallisepticum in serum-free, protein-free SP4 medium, serum-free, protein-free R2 medium, serum-free, protein-free mHF medium, serum-free, protein-free MBB medium, FRIIS medium, and Frey medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD.Figure 12 is a graph showing the growth rate of M. gallisepticum in serum-free, protein-free SP4 medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 13 is a graph showing the growth rate of M. gallisepticum in serum-free, protein-free R2 medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. The upper row shows poor growth, and the lower row shows good growth. Figure 14 is a graph showing the growth rate of M. synoviae in serum-free, protein-free SP4 medium, R2 medium, FRIIS medium, and Frey medium containing serum or supplemented with lipid, PVA, and MβCD. Figure 15 is a graph showing the growth rate of M. synoviae in serum-free, protein-free SP4 medium, serum-free, protein-free R2 medium, serum-free, protein-free mHF medium, and serum-free, protein-free MBB medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 16 is a graph showing the growth rate of S. chrysopicola in serum-free, protein-free SP4 medium, serum-free, protein-free R2 medium, serum-free, protein-free mHF medium, and serum-free, protein-free MBB medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 17 is a graph showing the growth rate of M. pneumoniae in serum-free, protein-free SP4 medium, serum-free, protein-free R2 medium, serum-free, protein-free mHF medium, and serum-free, protein-free MBB medium containing serum or supplemented with various concentrations of lipid, PVA, and MβCD. Figure 18 is a graph showing the growth rate of M. mycoides subcultured in SP4 medium (control), SP4-NT (control), SP4-LPC4 medium, and SP4-LPC4+SM:PC medium.

[0018] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0019] The present invention provides a serum-free, protein-free medium for culturing mycoplasma, comprising the following components: (a) a basal medium, (b) a lipid mixture containing cholesterol, palmitic acid, and oleic acid, (c) a polyvinyl alcohol-based resin, and (d) cyclodextrin.

[0020] In the present invention, "serum-free" means not only that serum is not supplemented, but also that purified serum-derived components (such as albumin and growth factors) are not supplemented.

[0021] In the present invention, "protein-free" means substantially free of protein, i.e., no protein is detected by commonly used protein detection means (e.g., Coomassie Brilliant Blue (CBB) staining, silver staining, etc.). Therefore, low-molecular-weight peptides (e.g., less than 10 kDa) that are not detectable by the above-mentioned detection means may be contained in the serum-free, protein-free medium of the present invention.

[0022] In the present invention, "mycoplasma" refers to bacteria generally classified into the class Mollicutes, commonly referred to as mycoplasma. Representative examples include bacteria of the genera Mycoplasma, Ureaplasma, Spiroplasma, and Acholeplasma. These genera include many pathogenic bacterial species for a wide range of organisms, including humans, livestock, crustaceans, and insects. Examples of such pathogenic bacterial species include Mycoplasma pneumoniae (hereinafter referred to as M. pneumoniae), which causes human mycoplasma pneumonia; Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum, which cause human non-chlamydial, non-gonococcal urethritis; and Mycoplasma gallisepticum, which causes chicken mycoplasmosis. gallisepticum (hereinafter, M. gallisepticum) and Mycoplasma synoviae (hereinafter, M. synoviae), which cause bovine mycoplasmosis; and Mycoplasma mycoides (hereinafter, M. mycoides) and Mycoplasma bovis, which cause bovine mycoplasmosis. Examples of bacterial species that infect insects or crustaceans include, but are not limited to, Spiroplasma chrysopicola (hereinafter, S. chrysopicola), which infects dipteran insects, and Spiroplasma eriocheiris (hereinafter, S. eriocheiris), which infects crustaceans. Mycoplasmas in the present invention may also include species, subspecies, or strains that are phylogenetically closely related to the above bacteria.

[0023] Component (a) is a basal medium. A "basal medium" refers to a medium containing only specified components essential for the growth of the cells to be cultured and containing no additives such as serum. Numerous basal media for culturing mycoplasma have already been established, and any known basal medium can be used for the medium of the present invention. Examples of such basal media include, but are not limited to, SP4 medium, mycoplasma broth-based medium, R2 medium, Hayflick medium, FRIIS medium, Frey medium, and modified media thereof. Component (a) can be any of the above, either alone or in combination. When two or more types are combined, the ratio between them can be arbitrary. Preferably, the basal medium (a) of the present invention is SP4 medium.

[0024] The basal medium (a) that can be used in the present invention is commercially available, and commercially available products can also be used. Specifically, for example, Frey mycoplasma broth base (Merck Millipore, F6797), Hayflick broth base (HiMedia, ME1885), Mycoplasma broth base (Thermo Fisher Scientific, CM0403B), etc. can be used. On the other hand, although media to which serum has already been added are commercially available (e.g., SP4-Z medium (ATCC, 2764), Remel SP4 Glucose Broth (Thermo Fisher Scientific, R112585), FRIIS Liquid Medium acc. EP (Merck Millipore, 146180), etc.), they should not be used as the basal medium (a) of the serum-free, protein-free medium of the present invention.

[0025] Component (b) is a lipid mixture containing cholesterol, palmitic acid, and oleic acid. In the present invention, "cholesterol," "palmitic acid," and "oleic acid" may be in the free form or in the ester form, but are preferably in the free form.

[0026] The lipid mixture (b) may contain cholesterol, palmitic acid, and oleic acid in any ratio, preferably 10:5:6 or 2:1:1, and the ratio of each component may be changed within a range of 1 / 2 to 4 times the above ratio. Thus, the ratio of cholesterol, palmitic acid, and oleic acid may be, for example, 40:5:6, 40:5:3, 10:20:6, 5:20:6, 10:2.5:24, or 10:5:24.

[0027] The lipid mixture (b) may consist solely of cholesterol, palmitic acid, and oleic acid, or may further contain other lipids that are commonly used as additives in culture media. Examples of such lipids include, but are not limited to, sphingomyelin and phosphatidylcholine. The content of such lipids may be, for example, 0 to 20 mg / L (final concentration in the culture medium).

[0028] The content of the lipid mixture (b) in the serum-free, protein-free medium of the present invention may be, for example, 5 to 120 mg / L, 6.5 to 100 mg / L, and preferably 21 to 45 mg / L. More specifically, the serum-free, protein-free medium of the present invention may contain, for example, 5 to 40 mg / L, preferably 10 to 20 mg / L, of cholesterol; 0.5 to 30 mg / L, preferably 5 to 10 mg / L, of palmitic acid; and 1 to 30 mg / L, preferably 6 to 15 mg / L, of oleic acid.

[0029] Component (c) is a polyvinyl alcohol-based resin. In the present invention, the "polyvinyl alcohol-based resin" refers to polyvinyl alcohol, a polyvinyl alcohol derivative, or a polyvinyl alcohol copolymer. Component (c) can be any of the above-mentioned resins, either alone or in combination of two or more. When two or more types are combined, the ratio between them may be any. Preferably, the polyvinyl alcohol-based resin (c) in the present invention is polyvinyl alcohol.

[0030] The polyvinyl alcohol-based resin (c) in the present invention may have any degree of polymerization and any degree of saponification. The average degree of polymerization may be, for example, 500 to 3500. The average degree of saponification may be, for example, 75 to 100 mol%. The degree of polymerization and the degree of saponification can be measured, for example, in accordance with JIS K 6726:1994. However, when a commercially available product is used as the polyvinyl alcohol-based resin (c) in the present invention, the values ​​indicated on the product may be used.

[0031] The polyvinyl alcohol-based resin (c) that can be used in the present invention is commercially available, and commercially available products can also be used. Specific examples that can be used include polyvinyl alcohol (Sigma-Aldrich, P8136) (average degree of polymerization 680 to 1600, average degree of saponification 87 to 90 mol%), polyvinyl alcohol (Fujifilm Wako Pure Chemical Industries, Ltd., 162-16325) (average degree of polymerization 900 to 1000, average degree of saponification 96 mol%), polyvinyl alcohol (Nacalai Tesque, Ltd., 15948-65) (average degree of polymerization 2000, average degree of saponification 86.5 to 89 mol%), and polyvinyl alcohol (Kanto Chemical, Ltd., 32784-00) (average degree of polymerization 2000, average degree of saponification 78 to 82 mol%).

[0032] The content of the polyvinyl alcohol resin (c) in the serum-free, protein-free medium of the present invention may be, for example, 0.2 to 5.0 w / v %, and preferably 1.0 to 3.0 w / v %, when the total mass of the serum-free, protein-free medium is taken as 100%.

[0033] Component (d) is a cyclodextrin. "Cyclodextrin" is a cyclic oligosaccharide composed of five or more glucose units linked by α-1,4 bonds. Examples of cyclodextrin (d) in the present invention include, but are not limited to, α-cyclodextrin composed of six glucose units, β-cyclodextrin composed of seven glucose units, γ-cyclodextrin composed of eight glucose units, and derivatives thereof. Cyclodextrin derivatives may have some or all of the hydroxyl groups in the glucose units chemically modified, and examples include, but are not limited to, methylated cyclodextrin, hydroxypropylated cyclodextrin, and acetylated cyclodextrin. Component (d) can be any of the above compounds selected alone or in combination of two or more, and when two or more compounds are combined, the ratio between them can be any.

[0034] The cyclodextrin (d) in the present invention is preferably a methylated cyclodextrin, and particularly preferably methyl-β-cyclodextrin. The methylated cyclodextrin that can be used in the present invention may be a partially methylated cyclodextrin in which the hydroxyl groups of the cyclodextrin are partially methylated, or a fully methylated cyclodextrin in which all the hydroxyl groups of the cyclodextrin are methylated. The methylation rate of the methylated cyclodextrin that can be used in the present invention may be, for example, 15 to 100%, preferably 50 to 70%. The methylation rate can be measured, for example, by gas chromatography-mass spectrometry (GC / MS). When a commercially available product is used as the cyclodextrin (d) in the present invention, the methylation rate may be the value indicated on the product.

[0035] The cyclodextrin (d) that can be used in the present invention is commercially available, and commercially available products can also be used. Specifically, for example, methyl-β-cyclodextrin (Fujifilm Wako Pure Chemical Industries, Ltd., 320-84252), methyl-β-cyclodextrin (Sigma-Aldrich, 779776 and C4555), methyl-β-cyclodextrin (Tokyo Chemical Industry, Ltd., M1356), methyl-β-cyclodextrin (Junsei Chemical, Ltd., 74148-1605), etc. can be used.

[0036] The content of cyclodextrin (d) in the serum-free, protein-free medium of the present invention may be, for example, 0.1 to 3.0 w / v %, and preferably 0.2 to 2.0 w / v %, when the total mass of the serum-free, protein-free medium is taken as 100%.

[0037] In addition to the above components, the serum-free, protein-free medium of the present invention can contain appropriate components used in conventional media, such as amino acids, salts, sugars, buffers, trace elements, antibiotics, and vitamins, within limits that do not impair the effects of the present invention.

[0038] The method for producing the serum-free, protein-free medium of the present invention is not particularly limited, and the medium can be produced by adding components (b) to (d) and other components to basal medium (a) and mixing them.

[0039] The serum-free, protein-free medium of the present invention is inexpensive and has excellent quality consistency because it does not contain serum or albumin, and is therefore useful for testing and research on mycoplasma.

[0040] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0041] <A. Reagents> The reagent information (reagent name, abbreviation, manufacturer, catalog number / product number, etc.) used in this example is as follows:

[0042] Table 1. List of reagents (The abbreviations in the table are as follows: Mfr: manufacturer, Cat.#: catalog number / product number, BD: Becton Dickinson, SFS: Thermo Fisher Scientific, Wako: Fujifilm Wako Pure Chemical Industries, Sigma: Sigma-Aldrich, MPB: MP Biomedicals)

[0043] <B. Stock Solutions> The following stock solutions were prepared. Unless otherwise specified, the stock solutions were filtered through a sterilizing grade filter (Millex-GV, 0.22 μm (Merck Millipore)) and stored at 4°C. Hereinafter, all "%" means w / v %. Cholesterol solution (0.5%): 50 mg of cholesterol was dissolved in 10 mL of ethanol. Palmitic acid solution (0.25%): 25 mg of cholesterol was dissolved in 10 mL of ethanol. Oleic acid solution (0.3%): 30 mg of cholesterol was dissolved in 10 mL of ethanol. Sphingomyelin solution (0.5%): 50 mg of sphingomyelin was dissolved in 10 mL of methanol. BSA solution (10%): 1 g of BSA was dissolved in 10 mL of Milli-Q water. Tween solution (10%): 1 g of Tween 20, 40, 60, or 80 was dissolved in 9 mL of Milli-Q water. Polyvinyl alcohol (PVA) solution (10%): 5 g of PVA was dissolved in 45 mL of Milli-Q water. Methyl-β-cyclodextrin (MβCD) solution (30%): 15 g of MβCD was dissolved in 50 mL of Milli-Q water. β-NAD solution (1%): 10 mg of β-NAD was dissolved in 1 mL of Milli-Q water. NaCl solution (0.45%): 2.25 g of NaCl was dissolved in 500 mL of Milli-Q water. Sterilized by filtration or autoclaving and stored at room temperature. L-glutamine solution (200 mM): 5.8 g of L-glutamine was dissolved in 200 mL of Milli-Q water. Sterilized by filtration and stored at -20°C. TC Yeastolate Solution (200 mM): 10 g of TC Yeastolate was dissolved in 500 mL of Milli-Q water. After sterilization by filtration or autoclaving, the solution was stored at 4°C. Penicillin G Solution (400,000 U / mL): 5.34 g of penicillin G (1500 units / mg) was dissolved in 20 mL of Milli-Q water. After sterilization by filtration, the solution was stored at -20°C. Ampicillin Solution (100 mg / mL): 10 g of ampicillin was dissolved in 100 mL of Milli-Q water. After sterilization by filtration, the solution was stored at -20°C. Phenol Red Solution (1%): 1 g of phenol red was dissolved in 100 mL of Milli-Q water. After sterilization by filtration, the solution was stored at room temperature or 4°C.Glucose solution (20%): 100 g of D(+)-glucose was dissolved in 500 mL of Milli-Q water. After sterilization by filtration or autoclaving, the solution was stored at 4°C. Sodium bicarbonate solution (7.5%): 15 g of sodium bicarbonate was dissolved in 200 mL of Milli-Q water. After sterilization by filtration or autoclaving, the solution was stored at 4°C.

[0044] <C. Culture Medium> A liquid medium (hereinafter also referred to simply as "medium") was prepared by the following procedure. A solid medium (hereinafter also referred to as "plate medium" or simply as "plate") was prepared by adding 1% agar to the liquid medium and allowing it to solidify at room temperature.

[0045] (1-1) SP4 Medium (Serum+) The following Part 1 and Part 2 were prepared and mixed in a ratio of Part 1:Part 2 = 3:2 to obtain SP4 Medium (Serum+). For Part 1, the reagents were dissolved in Milli-Q water, adjusted to pH 7.5 with 4 M KOH, sterilized by autoclave, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 3.5 g Mycoplasma Broth Base, 10 g Tryptone, 5.3 g Peptone, and 0 or 10 g Agar in 600 mL Milli-Q water (total volume 600 mL) Part 2: 25 mL Dextrose Solution, 50 mL 10x CMRL1066, 14.6 mL Sodium Bicarbonate Solution, 5 mL L-Glutamine Solution, 35 mL Fresh Yeast Extract, 100 mL TC Yeastolate Solution, 170 mL Fetal Bovine Serum (inactivated), 2.5 mL Penicillin G Solution, and 1.5 mL Phenol Red Solution (total volume 403.6 mL) Final Concentrations: 3.5 g / L Mycoplasma broth base, 10 g / L tryptone, 5.3 g / L peptone, 0 or 1% agar, 0.5 g / L glucose, 0.5x CMRL1066, 0.11% sodium bicarbonate, 1 mM L-glutamine, 0.525% Fresh yeast extract, 0.2% TC Yeastolate, 17% (v / v) fetal bovine serum (inactivated), 1,000 U / mL penicillin G, and 0.0015% phenol red

[0046] (1-2) SP4 medium (serum-) The following Part 1, Part 2, and Part 3 were prepared and mixed in a ratio of Part 1:Part 2:Part 3=1:2:2 to obtain SP4 medium (serum-). Parts 1 and 2 were prepared in the same manner as in (1-1) above. Components that differ from SP4 medium (serum+) are underlined. Part 1: 3.5 g Mycoplasma broth base, 10 g tryptone, 5.3 g peptone, and 0 or 10 g agar / 200 mL Milli-Q water (total volume 200 mL) Part 2: 25 mL glucose solution, 50 mL 10x CMRL1066, 14.6 mL sodium bicarbonate solution, 5 mL L-glutamine solution, 35 mL fresh yeast extract, 100 mL TC yeastolate solution, 170 mL NaCl solution, 2.5 mL penicillin G solution, and 1.5 mL phenol red solution (total volume 403.6 mL) Final concentrations: 3.5 g / L Mycoplasma broth base, 10 g / L tryptone, 5.3 g / L peptone, 0 or 1% agar, 0.5 g / L glucose, 0.5x CMRL1066, 0.11% sodium bicarbonate, 1 mM L-glutamine, 0.525% Fresh yeast extract, 0.2% TC Yeastolate, 0.0765% NaCl, 1,000 U / mL penicillin G, and 0.0015% phenol red

[0047] Part 3 was prepared with a different composition. Part 3 contained lipids, BSA, PVA, MβCD, surfactants, glycerol, or sugars added instead of serum. For Part 3, the reagents were mixed, sterilized by filtration, and then stored at 4°C. NT: Milli-Q water wo: 4 mL cholesterol, 4 mL palmitic acid, 4 mL oleic acid, and 20 mL BSA solution (final concentrations: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 2 g / L BSA) 3L: 4 mL cholesterol, 4 mL palmitic acid, and 4 mL oleic acid (final concentrations: 20 mg / L cholesterol, 10 mg / L palmitic acid, and 12 mg / L oleic acid) 3L half: 2 mL cholesterol, 2 mL palmitic acid, and 2 mL oleic acid (final concentrations: 10 mg / L cholesterol, 5 mg / L palmitic acid, and 6 mg / L oleic acid) 3L double: 40 mg / L cholesterol, 20 mg / L palmitic acid, and 24 mg / L oleic acid (final concentrations: LPC1: 3 L, 100 mL PVA solution and 66.7 mL MβCD solution (final concentrations: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, 1% PVA, and 2% MβCD) LPC2: 3 L, 300 mL PVA solution and 66.7 mL MβCD solution (final concentrations: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, 3% PVA, and 2% MβCD) LPC3: 3 L half, 300 mL PVA solution and 33.3 mL MβCD solution (final concentrations: LPC4: 3 L half, 100 mL PVA solution, and 33.3 mL MβCD solution (final concentrations: 10 mg / L cholesterol, 5 mg / L palmitic acid, 6 mg / L oleic acid, 1% PVA, and 1% MβCD)LPC5: 3 L half, 200 mL PVA solution, and 33.3 mL MβCD solution (final concentrations: 10 mg / L cholesterol, 5 mg / L palmitic acid, 6 mg / L oleic acid, 2% PVA, and 1% MβCD) LPC8: 3 L half, 100 mL PVA solution, and 13.3 mL MβCD solution (final concentrations: 10 mg / L cholesterol, 5 mg / L palmitic acid, 6 mg / L oleic acid, 2% PVA, and 0.4% MβCD) LPC9: 3 L half, 100 mL PVA solution, 33.3 mL MβCD solution, and 2 mL sphingomyelin solution (final concentrations: 10 mg / L cholesterol, 5 mg / L palmitic acid, 6 mg / L oleic acid, 1% PVA, 1% MβCD, and 10 mg / L sphingomyelin)

[0048] (2-1) R2 Medium (Serum+) The following Part 1 and Part 2 were prepared and mixed at a ratio of Part 1:Part 2 = 4:1 to obtain R2 Medium (Serum+). For Part 1, the reagents were dissolved in Milli-Q water, sterilized by autoclaving, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 25 g heart infusion broth and 80 g sucrose / 800 mL Milli-Q water (total volume 800 mL) Part 2: 60 mL newborn calf serum, 0.5 mL ampicillin, 2 mL phenol red, and 137.5 mL Milli-Q water (total volume 200 mL) Final concentrations: 25 g heart infusion broth, 80 g sucrose, 6% (v / v) newborn calf serum, 0.05 mg / mL ampicillin, and 0.002% phenol red

[0049] (2-2) R2 Medium (Serum-) The following Parts 1 and 2, and Part 3 prepared in (1-2) above, were used. R2 medium (serum-) was obtained by mixing Part 1:Part 2:Part 3 in a ratio of 2:1:2. Parts 1 and 2 were prepared in the same manner as in (2-1) above. Components that differ from R2 medium (serum+) are underlined. Part 1: 25 g heart infusion broth and 80 g sucrose / 400 mL Milli-Q water (total volume 400 mL) Part 2: 60 mL NaCl solution, 0.5 mL ampicillin, 2 mL phenol red, and 137.5 mL Milli-Q water (total volume 200 mL) Final concentrations: 25 g heart infusion broth, 80 g sucrose, 0.027% NaCl, 0.05 mg / mL ampicillin, and 0.002% phenol red

[0050] (3-1) Modified Hayflick Medium (Serum+) The following Part 1 and Part 2 were prepared and mixed in a ratio of Part 1:Part 2 = 3:2 to obtain modified Hayflick (mHF) medium (Serum+). For Part 1, the reagents were dissolved in Milli-Q water, sterilized by autoclave, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 21 g mycoplasma broth base and 5 g glucose / 600 mL Milli-Q water (total volume 600 mL) Part 2: 200 mL heat-inactivated horse serum, 100 mL fresh yeast extract, 2 mL phenol red solution, and 98 mL Milli-Q water (total volume 400 mL) Final concentrations: 21 g / L mycoplasma broth base, 5 g / L glucose, 20% (v / v) heat-inactivated horse serum, 1.5% fresh yeast extract, and 0.002% phenol red

[0051] (3-2) Modified Hayflick Medium (Serum-) The following Parts 1 and 2, and Part 3 prepared in (1-2) above, were used. mHF medium (Serum-) was obtained by mixing Part 1:Part 2:Part 3 in a ratio of 1:2:2. Parts 1 and 2 were prepared in the same manner as in (3-1) above. Components that differ from mHF medium (Serum+) are underlined. Part 1: 21 g mycoplasma broth base and 5 g glucose / 200 mL Milli-Q water (total volume 200 mL) Part 2: 200 mL NaCl solution, 100 mL fresh yeast extract, 2 mL phenol red solution, and 98 mL Milli-Q water (total volume 400 mL) Final concentrations: 21 g / L mycoplasma broth base, 5 g / L glucose, 0.09% NaCl, 1.5% fresh yeast extract, and 0.002% phenol red

[0052] (4-1) Mycoplasma Broth-Based Medium (Serum+) The following Part 1 and Part 2 were prepared and mixed at a ratio of Part 1:Part 2 = 4:1 to obtain mycoplasma broth-based (MBB) medium (Serum+). For Part 1, the reagents were dissolved in Milli-Q water, sterilized by autoclave, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 20 g Mycoplasma broth base / 800 mL Milli-Q water (total volume 800 mL) Part 2: 100 mL heat-inactivated horse serum, 50 mL fresh yeast extract, 2.5 mL penicillin G solution, 2 mL phenol red solution, and 45.5 mL Milli-Q water (total volume 200 mL) Final concentrations: 20 g / L Mycoplasma broth base, 10% (v / v) heat-inactivated horse serum, 0.75% fresh yeast extract, 1,000 U / mL penicillin G, and 0.002% phenol red

[0053] (4-2) Mycoplasma Broth-Based Medium (Serum-) The following Parts 1 and 2, and Part 3 prepared in (1-2) above, were used. MBB medium (Serum-) was obtained by mixing Part 1:Part 2:Part 3 in a ratio of 2:1:2. Parts 1 and 2 were prepared in the same manner as in (4-1) above. Components that differ from MBB medium (Serum+) are underlined. Part 1: 20 g Mycoplasma broth base / 400 mL Milli-Q water (total volume 800 mL) Part 2: 100 mL NaCl solution, 50 mL Fresh yeast extract, 2.5 mL penicillin G solution, 2 mL phenol red solution, and 45.5 mL Milli-Q water (total volume 200 mL) Final concentrations: 20 g / L Mycoplasma broth base, 0.045% NaCl, 0.75% Fresh yeast extract, 1,000 U / mL penicillin G, and 0.002% phenol red

[0054] (5-1) FRIIS Medium (Serum+) The following Part 1 and Part 2 were prepared and mixed in a ratio of Part 1:Part 2 = 6:4 to obtain FRIIS Medium (Serum+). For Part 1, the reagents were dissolved in Milli-Q water, sterilized by autoclaving, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 2.1 g heart infusion broth, 4.5 g mycoplasma broth base, 6.2 g yeast extract, 36 mg Na ２ HPO ４ , 29 mg KH ２ P.O. ４ , 68 mg CaCl ２ , 3.9g NaCl, 194mg KCl, 49mg MgCl ２ , and 49 mg MgSO ２ Part 1: 200 mL heat inactivated horse serum, 2 mL phenol red solution, and 198 mL Milli-Q water (total volume 400 mL) Final concentrations: 2.1 g / L heart infusion broth, 4.5 g / L mycoplasma broth base, 6.2 g / L yeast extract, 36 mg / L Na ２ HPO ４, 29 mg / L KH ２ P.O. ４ , 68mg / L CaCl ２ , 3.9g / L NaCl, 194mg / L KCl, 49mg / L MgCl ２ , and 49 mg / L MgSO ２ , 20% heat-inactivated horse serum, and 0.002% phenol red

[0055] (5-2) FRIIS medium (serum-) The following Parts 1 and 2, and Part 3 prepared in (1-2) above, were used. FRIIS medium (serum-) was obtained by mixing Part 1: Part 2: Part 3 in a ratio of 2:4:4. Parts 1 and 2 were prepared in the same manner as in (5-1) above. Components that differ from FRIIS medium (serum+) are underlined. Part 1: 2.1 g heart infusion broth, 4.5 g mycoplasma broth base, 6.2 g yeast extract, 36 mg Na ２ HPO ４ , 29 mg KH ２ P.O. ４ , 68 mg CaCl ２ , 3.9g NaCl, 194mg KCl, 49mg MgCl ２ , and 49 mg MgSO ２ Part 1: 200 mL NaCl solution, 2 mL phenol red solution, and 198 mL Milli-Q water (total volume 400 mL) Final concentrations: 2.1 g / L Heart Infusion Broth, 4.5 g / L Mycoplasma Broth Base, 6.2 g / L Yeast Extract, 36 mg / L Na ２ HPO ４ , 29 mg / L KH ２ P.O. ４ , 68mg / L CaCl ２ , 3.9g / L NaCl, 194mg / L KCl, 49mg / L MgCl ２ , and 49 mg / L MgSO ４ , 0.09% NaCl, and 0.002% phenol red

[0056] (6-1) Frey medium (serum+) The following Part 1 and Part 2 were prepared and mixed in a ratio of Part 1:Part 2 = 6:4 to obtain Frey medium (serum+). For Part 1, the reagents were dissolved in Milli-Q water, sterilized by autoclaving, and stored at 4°C. For Part 2, the reagents were mixed, sterilized by filtration, and stored at 4°C. Part 1: 7.5 g tryptone, 2.5 g soytone, 5.0 g yeast extract, 5.0 g NaCl, 0.4 g KCl, 0.2 g MgSO ４ ・7H ２ O, 1.6g Na ２ HPO ４ , and 0.1 g KH ２ P.O. ４ Part 1: 100 mL heat-inactivated horse serum, 10 mL β-NAD solution, 10 mL L-cysteine ​​solution, 2 mL phenol red solution, and 280 mL Milli-Q water (total volume 400 mL) Final concentrations: 7.5 g / L tryptone, 2.5 g / L soytone, 5.0 g / L yeast extract, 5.0 g / L NaCl, 0.4 g / L KCl, 0.2 g / L MgSO ４ ・7H ２ O, 1.6g / L Na ２ HPO ４ , 0.1g / L KH ２ P.O. ４ , 10% heat-inactivated horse serum, 0.01% β-NAD, 0.01% L-cysteine, and 0.002% phenol red

[0057] (6-2) Frey medium (serum-) The following Parts 1 and 2, and Part 3 prepared in (1-2) above, were used. FRIIS medium (serum-) was obtained by mixing Part 1: Part 2: Part 3 in a ratio of 2:4:4. Parts 1 and 2 were prepared in the same manner as in (6-1) above. Components that differ from FRIIS medium (serum+) are underlined. Part 1: 7.5 g tryptone, 2.5 g soytone, 5.0 g yeast extract, 5.0 g NaCl, 0.4 g KCl, 0.2 g MgSO ４ ・7H ２ O, 1.6g Na ２ HPO４ , and 0.1 g KH ２ P.O. ４ Part 1: 200 mL NaCl solution, 10 mL β-NAD solution, 10 mL L-cysteine ​​solution, 2 mL phenol red solution, and 280 mL Milli-Q water (total volume 400 mL) Final concentrations: 7.5 g / L tryptone, 2.5 g / L soytone, 5.0 g / L yeast extract, 5.0 g / L NaCl, 0.4 g / L KCl, 0.2 g / L MgSO ４ ・7H ２ O, 1.6g / L Na ２ HPO ４ , 0.1g / L KH ２ P.O. ４ , 0.09% NaCl, 0.01% β-NAD, 0.01% L-cysteine, and 0.002% phenol red

[0058] In the following examples, SP4 medium (serum+) will be referred to simply as "SP4," and SP4 medium (serum-) will be referred to in the format of "SP4-Part 3 name." For example, SP4 medium (serum-) using LPC1 as Part 3 will be referred to as "SP4-LPC1." The same applies to other media; for example, R2 medium (serum+) will be referred to simply as "R2," and R2 medium (serum-) using LPC1 as Part 3 will be referred to as "R2-LPC1."

[0059] <D. Strains> Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma pneumoniae, Spiroplasma eriocheilis, and Spiroplasma chrysopicola were obtained from the American Type Culture Collection (ATCC) or provided by a collaborator (Professor Masato Miyata, Osaka Municipal University).

[0060] <E. Evaluation Method> (i) Culture in Liquid Medium A bacterial suspension frozen and stored at -80°C was thawed. 50 μL of the bacterial suspension was added to fresh SP4 medium (500 μL) and cultured until the early stationary phase. S. eriochaelis and S. chrysopicola were cultured in a constant-temperature shaking incubator (Bioshacker BR-43FL, Taitec) at a set temperature of 30°C, while the other strains were cultured at a set temperature of 37°C. As the bacteria grew, the medium became acidic, and the color of the medium (phenol red) changed from red to orange and then to yellow. The stationary phase was marked by an orange to yellow color, and this was used as an indicator to stop the culture. The bacterial suspension was centrifuged at 13,000 × g for 1 minute, the supernatant was removed, and 500 μL of serum-free, protein-free medium (NT) was added to suspend the bacteria. The serum-free, protein-free medium (NT) used corresponded to the medium used for subsequent evaluation culture. This inoculum was inoculated into various media at 1-10% by volume, and growth was evaluated. The inoculum was 5-10% for M. gallicepricum and M. pneumoniae, and 1% for other strains. The number of days required for the medium to turn orange or yellow after inoculation was measured as the growth time. Growth was also evaluated by measuring absorbance at 430 nm and 560 nm using a 96-well plate (FALCON 353072) and a plate reader (TECAN SPARK 10M). A method for measuring mycoplasma growth based on the ratio of absorbance at 430 nm and 560 nm (OD430 / 560) has already been reported (Yus et al., Science, 2009; 326(5957): 1263-1268 and Mariscal et al., ACS Synth. Biol., 2018; 7(6): 1538-1552).

[0061] (ii) Cultivation on solid media Bacteria cultured using SP4 were serially diluted using SP4-NT. The dilution series (10 μL each) was applied to plates prepared from various liquid media and cultured statically at 37°C. After 2 to 6 days, when the colonies had grown sufficiently, the colony shapes were observed using a stereomicroscope (M125 C, Leica) and the number of colonies was counted.

[0062] (iii) Cultivation of frozen-thawed bacterial cells (liquid medium) The bacterial solutions after culturing in various media were frozen and stored at −80° C. After two weeks, the frozen stocks were thawed and inoculated into fresh liquid media at 1 to 10% by volume, and the growth of the frozen-thawed bacterial cells was evaluated.

[0063] <Preliminary Experiment 1: Lipids> First, the growth rates of Mycoplasma mycoides subsp. capri (hereinafter simply referred to as "M. mycoides") in SP4-3L, SP4-3L half, and SP4-3L double prepared in (1-1) above were compared using the procedure in Section E. The growth rate was calculated as the reciprocal of the growth time (days). M. mycoides grew best in SP4-3L half (growth rate: 0.15), and also in SP4-3L (growth rate: 0.1), but growth was significantly worse than in SP4. No growth was observed in SP4-3L double. Furthermore, M. mycoides grew slightly (growth rate: 0.1) in media supplemented with low concentrations of palmitic acid (0.5 or 1 mg / L).

[0064] Preliminary Experiment 2: Lipid + BSA. SP4-3L was supplemented with 0.1 to 2 g / L of BSA, and the growth of M. mycoides was compared. The higher the BSA concentration, the better the growth. However, even the growth in SP4-wo (SP4-3L + 2 g / L BSA) (growth rate: 0.3) was poorer than that in SP4 (growth rate: 1.0).

[0065] Preliminary Experiment 3: Lipid + Detergent, Glycerol, or Sugar. SP4-3L media were prepared by adding a surfactant (polysorbate (Tween)), glycerol, or sugar instead of BSA (Tween 1%, 0.1%, or 0.05%, glycerol 1% or 0.1%, sucrose 0.5%, galactose 0.5%, sodium acetate 0.5%, glycerol 0.5% + sucrose 0.5%, and glycerol 0.5% + galactose 0.5%), and the growth rates of M. mycoides were compared. The final concentrations of the additives in each medium were as follows: Tween_1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 1% Tween 20, 40, 60, or 80. Tween_0.1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.1% Tween 20, 40, 60, or 80. Tween_0.05%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.05% Tween 20, 40, 60, or 80. Glycerol_1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 1% glycerol. Glycerol_0.1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.1% glycerol. Sucrose 0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.5% sucrose. Galactose 0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.5% galactose. Na-acetate 0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.5% sodium acetate. Glycerol 0.5% + Sucrose 0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, 0.5% glycerol, and 0.5% sucrose. Glycerol 0.5% + Galactose 0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, 0.5% glycerol, and 0.5% galactose

[0066] As a result, M. mycoides did not grow in any of these media. As described above, M. mycoides grew in SP4-3L, suggesting that surfactants, glycerol, or sugars have a negative effect on the growth of M. mycoides.

[0067] Preliminary Experiment 4: Lipid + Polyvinyl Alcohol (PVA) Media were prepared by adding 0.2 to 3% PVA to SP4-3L half instead of BSA (PVA_0.2%, PVA_0.5%, PVA_1%, PVA_2%, and PVA_3%), and the growth rates of M. mycoides were compared. The final concentrations of the additives in each medium were as follows: PVA_0.2%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.2% PVA. PVA_0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.5% PVA. PVA_1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 1% PVA. PVA_2%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 2% PVA. PVA_3%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 3% PVA.

[0068] As a result, M. mycoides grew slightly in 0.2%, 0.5%, and 1% PVA (growth rate: 0.1), but grew slightly better in 2% and 3% PVA (growth rate: 0.15).

[0069] <Preliminary Experiment 5: Lipid + Methyl-β-cyclodextrin (MβCD)> Media were prepared by adding 0.2 to 3% MβCD to SP4-3L half instead of BSA (mbCD_0.2%, mbCD_0.5%, mbCD_1%, mbCD_2%, and mbCD_3%; see (1-1) above), and the growth rates of M. mycoides were compared. The final concentrations of the additives in each medium were as follows: mbCD_0.2%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.2% MβCD; mbCD_0.5%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 0.5% MβCD; mbCD_1%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 1% MβCD; mbCD_2%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 2% MβCD; mbCD_3%: 20 mg / L cholesterol, 10 mg / L palmitic acid, 12 mg / L oleic acid, and 3% MβCD

[0070] As a result, M. mycoides grew in mbCD_0.2%, mbCD_0.5%, and mbCD_1% (growth rates: 0.18, 0.2, and 0.5), but did not grow in mbCD_2% or mbCD-3%.

[0071] The results of preliminary experiments 1 to 5 confirmed that M. mycoides could grow in serum-free, protein-free SP4 medium supplemented with three types of lipids (cholesterol, palmitic acid, and oleic acid) and BSA, PVA, or MβCD. Based on these results, the concentrations of lipids, BSA, PVA, and MβCD were optimized in the following examples.

[0072] Example 1: M. mycoides / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium) Media containing various concentrations of PVA and MβCD added to SP4-3L or SP4-3L half were prepared, and the growth rates of M. mycoides were compared according to the procedure described in Section E. The results are shown in Figure 1. The range of PVA and MβCD concentrations suitable for growing M. mycoides differed between the addition of both in combination and the addition of either alone. The relationship between lipid and MβCD concentrations is shown in Figure 2. In the figure, open circles indicate concentrations at which growth was favorable, black circles indicate concentrations at which growth was only achieved in the presence of high concentrations of PVA, gray circles indicate concentrations at which growth was poor, and x indicates concentrations at which little growth was achieved.

[0073] Next, lipid concentrations were examined. Figure 3 shows the growth rates of M. mycoides in SP4-wo media with varying lipid concentrations, and Figure 4 shows the growth rates of M. mycoides in SP4-LPC4 media with varying lipid concentrations. Good growth was observed in 3 L (20 mg / L cholesterol, 10 mg / L palmitic acid, and 12 mg / L oleic acid) and 3 L half (10 mg / L cholesterol, 5 mg / L palmitic acid, and 6 mg / L oleic acid). The media that showed good growth (SP4-LPC1, SP4-LPC2, SP4-LPC3, SP4-LPC4, and SP4-LPC5) were used in subsequent studies.

[0074] The growth rates of M. mycoides in SP4-LPC1, SP4-LPC2, SP4-LPC3, SP4-LPC4, and SP4-LPC5 were measured based on OD430 / 560. SP4, SP4-wo, SP4-NT, SP4-3L half + 3% PVA, and SP4-3L half + 1% MβCD were used as control media.

[0075] The results are shown in Figures 5 and 6. In the figures, "3LH + 3% PVA" indicates 3L half + 3% PVA, and "3LH + 1% mbCD" indicates 3L half + 1% MβCD. Good growth was not observed in media supplemented with only lipid and PVA, or only lipid and MβCD (Figure 5). On the other hand, in SP4-LPC1, SP4-LPC2, SP4-LPC3, SP4-LPC4, and SP4-LPC5 supplemented with lipid, PVA, and MβCD, the initiation of growth was slightly delayed compared to SP4, but the growth rate (slope of the growth curve) was equivalent to that of SP4.

[0076] These results demonstrate that serum-free, protein-free SP4 medium supplemented with lipids, PVA, and MβCD can be used to grow M. mycoides.

[0077] Example 2: M. mycoides / Lipid + PVA + MβCD (Serum-Free, Protein-Free R2 Medium, mHF Medium, and MBB Medium) The growth of M. mycoides was similarly tested using R2 medium, mHF medium, or MBB medium instead of SP4 as the basal medium. The results are shown in Figure 7. M. mycoides was also able to grow in R2-LPC1 to LPC3 and mHF-LPC1 to LPC3, but growth was slower than in SP4-LPC1 to 3. M. mycoides did not grow when MBB medium was used as the basal medium. Particularly good growth was observed in SP4-LPC9, which further contained sphingomyelin in addition to 3 L half, PVA, and MβCD.

[0078] Example 3: M. mycoides / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Plate Medium) SP4, SP4-wo, and SP4-LPC1 to 5 plates were prepared and tested for colony formation by M. mycoides. The results are shown in FIG. 8. In the figure, the letters (a, ab, b, and c) above the graph indicate the results of significant tests (ANOVA and T-test), and indicate that significant differences were observed between different test groups (p<0.05). Colonies formed on all plates, and all had colony shapes characteristic of mycoplasma. The most colonies formed were in SP4-LPC3.

[0079] Example 4: Cryopreservation (M. mycoides / serum-free, protein-free SP4 medium) M. mycoides was cultured in SP4 or SP4-LPC1 to 5, then stored at -80°C, and a recovery test from freezing was performed after 2 weeks. The results are shown in Figure 9. M. mycoides recovered from freezing except when cultured in SP4-LPC1 before and after storage. In particular, when cultured in SP4-LPC4 or SP4-LPC5 before and after storage, M. mycoides grew at a rate equivalent to that when cultured in SP4 before and after storage.

[0080] Example 5: Proteins in Culture Media (Serum-Free, Protein-Free SP4 Medium) SP4, SP4-wo, SP4-3L, SP4-NT, and SP4-LPC1 to SP4-LPC5 were subjected to SDS-PAGE and CBB staining to detect proteins contained in each medium. The results are shown in Figure 10. Strong bands were detected in SP4 containing serum and SP4-wo containing BSA, but no visible bands were detected in SP4-LPC1 to SP4-LPC5. These results suggest that SP4-LPC1 to SP4-LPC5 are substantially protein-free and are useful for research on mycoplasma proteins.

[0081] Example 6: M. gallisepticum / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium, R2 Medium, mHF Medium, MBB Medium, FRIIS Medium, and Frey Medium) Growth of M. gallisepticum was tested using SP4 medium, R2 medium, mHF medium, MBB medium, FRIIS medium, or Frey medium as the basal medium according to the procedure in Section E. The results are shown in Figure 11. Growth of M. gallisepticum was observed in LPC medium using R2 medium, FRIIS medium, or Frey medium as the basal medium. On the other hand, M. gallisepticum did not grow in LPC medium using SP4 medium, mHF medium, or MBB medium as the basal medium.

[0082] The growth rates of M. gallisepticum measured based on OD430 / 560 are shown in Figures 12 and 13. M. gallisepticum did not grow in LPC medium using SP4 medium as the basal medium (Figure 12). In LPC2-5 media using R2 medium as the basal medium, growth of M. gallisepticum was observed that was comparable to that in serum-containing R2 medium (Figure 13, lower panel). These results were consistent with those in Figure 11.

[0083] Example 7: M. synoviae / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium, R2 Medium, FRIIS Medium, and Frey Medium) Growth of M. synoviae was tested according to the procedure described in Section E using LPC medium with SP4 medium, R2 medium, FRIIS medium, or Frey medium as the basal medium. β-NAD (final concentration: 0.01%), which is essential for M. synoviae growth, was also added. The results are shown in Figure 14. Good growth of M. synoviae was observed in LPC medium + β-NAD regardless of whether SP4 medium, R2 medium, FRIIS medium, or Frey medium was used as the basal medium.

[0084] Example 8: S. chrysopicola / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium, R2 Medium, mHF Medium, and MBB Medium) Growth of S. chrysopicola was tested using LPC medium with SP4 medium, R2 medium, mHF medium, or MBB medium as the basal medium according to the procedure in Section E. The results are shown in Figure 15. S. chrysopicola grew in serum-containing SP4 medium, R2 medium, or mHF medium within 1 to 2 days. Regarding serum-free, protein-free media, growth of S. chrysopicola was observed in LPC medium with SP4 medium or R2 medium as the basal medium, but S. chrysopicola did not grow in LPC medium with mHF medium or MBB medium as the basal medium. Good growth of S. chrysopicola was observed in SP4-LPC9, which further contained sphingomyelin.

[0085] Example 9: S. eriocheilis / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium, R2 Medium, mHF Medium, and MBB Medium) Growth of S. eriocheilis was tested according to the procedure described in Section E using LPC medium with SP4 medium, R2 medium, mHF medium, or MBB medium as the basal medium. The results are shown in Figure 16. S. eriocheilis grew in serum-containing SP4 medium, R2 medium, or mHF medium within 1 to 2 days. In serum-free, protein-free media, growth of S. eriocheilis was observed in R2-LPC1 and R2-LPC2, but the growth rate was approximately half that of serum-containing R2 medium. Good growth of S. eriocheilis was observed in SP4-LPC9.

[0086] Example 10: M. pneumoniae / Lipid + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium, R2 Medium, mHF Medium, and MBB Medium) The growth of M. pneumoniae was tested by the procedure in Section E using LPC medium with SP4 medium, R2 medium, mHF medium, or MBB medium as the basal medium. The results are shown in Figure 17. In SP4-LPC1 to 3, M. pneumoniae grew equally well as when cultured in serum-containing SP4 medium, R2 medium, or mHF medium.

[0087] The results of Examples 1 to 10 are summarized in Tables 2 to 6. Adding a combination of cholesterol, palmitic acid, oleic acid, PVA, and cyclodextrin to a serum-free, protein-free medium enabled the growth of all strains. A: Highly effective, B: Effective, C: Less effective, NT: Not tested.

[0088] Table 2. Cholesterol levels and effects on proliferation

[0089] Table 3. Concentration of palmitic acid and its effect on growth

[0090] Table 4. Concentration of oleic acid and its effect on growth

[0091] Table 5. Concentration of polyvinyl alcohol and its effect on proliferation

[0092] Table 6. Cyclodextrin concentrations and effects on growth

[0093] Example 11: M. mycoides / Lipids (5 Types) + PVA + MβCD (Serum-Free, Protein-Free SP4 Medium) SP4-LPC4, which was confirmed to grow well for M. mycoides in Example 1, was supplemented with sphingomyelin (SM) (2.5 mg / L) and phosphatidylcholine (PC) (1.25 mg / L) to prepare a medium called SP4-LPC4+SM:PC. M. mycoides was cultured using SP4, SP4-NT, SP4-LPC4, or SP4-LPC4+SM:PC according to the procedure described in Section E. When the medium turned orange or yellow, fresh medium was inoculated at 1% by volume, and the culture was passaged. Subculture was repeated three times, and the growth rates were compared according to the procedure described in Section E. The growth rate was calculated as the reciprocal of the growth time (days).

[0094] The results are shown in Figure 18. In the SP4-LPC4 medium, the proliferation rate decreased with repeated passage. On the other hand, in the SP4-LPC4+SM:PC medium, the proliferation rate did not decrease even with repeated passage. Without wishing to be bound by a particular theory, it was suggested that sphingomyelin and phosphatidylcholine are major lipid components of mycoplasma cell membranes, and that lipid metabolism affected the proliferation rate.

Claims

1. A serum-free, protein-free medium for culturing mycoplasma, comprising the following components: (a) a basal medium; (b) a lipid mixture containing cholesterol, palmitic acid, and oleic acid; (c) a polyvinyl alcohol-based resin; and (d) cyclodextrin.

2. The serum-free, protein-free medium according to claim 1, wherein the content of component (b) in the serum-free, protein-free medium is 5 to 120 mg / L.

3. The serum-free, protein-free medium according to claim 1 or 2, wherein the content of component (c) in the serum-free, protein-free medium is 0.2 to 3.0 w / v %.

4. A serum-free, protein-free medium according to any one of claims 1 to 3, wherein the content of component (d) in the serum-free, protein-free medium is 0.2 to 2.0 w / v%.

5. A serum-free, protein-free medium according to any one of claims 1 to 4, wherein the polyvinyl alcohol-based resin is polyvinyl alcohol.

6. The serum-free, protein-free medium according to any one of claims 1 to 5, wherein the cyclodextrin is a methylated cyclodextrin.

7. The serum-free, protein-free medium according to any one of claims 1 to 6, wherein the basal medium is SP4 medium.