Medium for culturing insect cells

Abstract

To provide a medium that allows culturing of diverse insect cells.SOLUTION: A medium for culturing insect cells, containing trehalose in an amount of 10-20 g / L.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a medium for culturing insect cells. [Background technology]

[0002] Insect culture cells are used as a useful tool in many fields, such as gene function analysis, protein expression, insect microbiology research, drug development, etc. In addition, insects have the largest number of species on Earth (1 million), and analyzing such a wide variety of organisms and their physiological phenomena holds great promise for the development of new biological resources and more useful tools.

[0003] However, even though they are generally referred to as insect cell cultures, their morphology and properties vary depending on the insect and tissue from which they are derived. Therefore, in order to analyze the physiological phenomena of each insect species more accurately and in detail, cultured cells derived from the insect being studied are required. However, a method suitable for culturing a wide variety of insect cells has not yet been established. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Chinese Patent Application Publication No. 102776148 [Non-patent literature]

[0005] [Non-Patent Document 1] Imogene Schneider, J Exp Zool, 1964, Volume 156, Issue 1, Pages 91-103 [Non-patent document 2] Technical Resources-Media Formulations 11720-Schneider's Drosophila Medium, [online], Thermo Fisher Scientific, Internet <URL:https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.124.html> Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a medium that enables the cultivation of various insect cells. [Means for solving the problem]

[0007] As a result of extensive research to achieve the above-mentioned object, the present inventors have found that cells collected from multiple species of insects (bees, whiteflies, aphids, and stink bugs) can be cultured using a medium containing a high concentration of trehalose (approximately 17 g / L). Specifically, while most insect cells survive for approximately two weeks under existing culture conditions, the use of a medium containing high concentrations of trehalose has enabled the production of insect cells with a survival period of two years or more. Furthermore, for insects of the order Hemiptera, for which the establishment of cultured cells has not been reported to date, the use of a medium containing high concentrations of trehalose has not only enabled survival for one year or more, but also cell proliferation, making it possible to establish cultured cells, leading to the completion of the present invention.

[0008] That is, the present invention provides the following aspects.

[0009] [1] A medium for culturing insect cells containing 4 to 35 g / L of trehalose.

[0010] [2] A medium for culturing insect cells containing 10-20 g / L of trehalose.

[0011] [3] The medium according to [1] or [2], wherein the culture is a primary culture.

[0012] [4] A method for producing cultured insect cells, comprising the step of culturing cells collected from an insect in the medium described in [1].

[0013] [5] The method according to [4], wherein the cultured insect cells are primary cultured cells.

[0014] Although many existing media use glucose or sucrose as the main sugar, media containing trehalose are also known (Non-Patent Documents 1 and 2, Patent Document 1). However, the trehalose concentration is low (Non-Patent Documents 1 and 2: 2 g / L, Patent Document 1: 0.5 to 3 g / L), suggesting that maintaining cells in such media tends to be difficult, as will be shown in the Examples below. [Effects of the Invention]

[0015] According to the present invention, it becomes possible to culture a variety of insect cells. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a micrograph showing the results of observing cells derived from European honeybee embryos cultured for 11 months in the medium of the present invention (MIPC-50: medium containing 17115 mg / L trehalose). [Figure 2] 1 is a micrograph showing the results of observing cells derived from Bemisia tabaci embryos cultured for 6 months in the medium of the present invention (MIPC-50). [Figure 3] 1 is a micrograph showing the results of observing cells derived from the embryos of the pea aphid after culturing them for 4 months in the medium of the present invention (MIPC-30: medium containing 16,477 mg / L trehalose). [Figure 4] 1 is a micrograph showing the results of observing cells derived from the embryos of the German-winged green stink bug after culturing them for 17 months in the medium of the present invention (MIPC-50). DETAILED DESCRIPTION OF THE INVENTION

[0017] <Culture medium> The present invention relates to a medium for culturing insect cells, which contains a high concentration of trehalose.

[0018] In the present invention, "trehalose" refers to a disaccharide formed by 1,1-glycosidic bonds between glucose units. More specifically, examples include α,α-trehalose, a disaccharide formed by 1,1-glycosidic bonds between two α-glucose units; α,β-trehalose, a disaccharide formed by 1,1-glycosidic bonds between an α-glucose and a β-glucose; and β,β-trehalose, a disaccharide formed by 1,1-glycosidic bonds between two β-glucose units. Of these, α,α-trehalose is preferred. Trehalose according to the present invention may also include derivatives thereof. Examples of such trehalose derivatives include glycosyltrehaloses in which one or more sugar units are bound to trehalose, more specifically glucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose, etc. Furthermore, the trehalose of the present invention also includes those that form salts (e.g., acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; metal salts such as sodium salt, potassium salt, and calcium salt; ammonium salt; and alkylammonium salt), hydrates (e.g., trehalose dihydrate), and solvates. Those skilled in the art can prepare such trehalose by any known method, such as chemical synthesis, microbial production, or enzymatic production. Alternatively, commercially available trehalose can be used.

[0019] The trehalose concentration (high concentration) in the medium of the present invention is preferably 4000 mg / mL or more, 5000 mg / L or more (e.g., 5135 mg / L (15 mM) or more), more preferably 6000 mg / L or more (e.g., 6846 mg / L (20 mM) or more), even more preferably 8000 mg / L or more (e.g., 8558 mg / L (25 mM) or more), more preferably 10000 mg / L or more, even more preferably 13000 mg / L or more, more preferably 15000 mg / L or more, even more preferably 16000 mg / L or more, and even more preferably 17000 mg / L or more (e.g., 17115 mg / L (50 mM) or more). Furthermore, it is preferably 35,000 mg / L or less (e.g., 34,230 mg / L (100 mM) or less), more preferably 30,000 mg / L or less (e.g., 25,763 ng / L (75 mM) or less), more preferably 25,000 mg / L or less, and even more preferably 20,000 mg / L or less (e.g., 17,115 mg / L (50 mM) or less). More specifically, the concentration of trehalose in the medium of the present invention is preferably 4000 to 35000 mg / L (e.g., 11.5 to 100 mM), more preferably 5000 to 35000 mg / L (e.g., 15 to 100 mM), even more preferably 6000 to 35000 mg / L (e.g., 20 to 100 mM), even more preferably 8000 to 30000 mg / L (e.g., 25 to 75 mM), and more preferably 10000 to 20000 mg / L (e.g., 30 to 50 mM). These weights are based on the amount of trehalose (C 12 H 22 O 11 When the above-mentioned trehalose derivatives, salts, hydrates, or solvates are contained in the medium of the present invention, it should be understood that the amounts are naturally calculated according to the molecular weight of each compound.

[0020] The medium of the present invention may contain other sugars in addition to the trehalose. Examples of such "other sugars" include glucose, fructose, sucrose, and maltose. Like the trehalose, these other sugars may be in the form of derivatives, salts, hydrates, or solvates. In addition, in the medium of the present invention, the glucose concentration is preferably 800 to 2,000 mg / L, more preferably 800 to 1,300 mg / L. The sucrose concentration is preferably 500 to 20,000 mg / L, more preferably 500 to 1,000 mg / L. The maltose concentration is preferably 300 to 500 mg / L.

[0021] The medium of the present invention may also contain amino acids. Examples of such "amino acids" include α-alanine, β-alanine, L-arginine-HCl, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-cystine, L-isoleucine, L-leucine, L-lysine-HCl, L-methionine, L-phenylalanine, L-proline, DL-serine, L-threonine, L-tryptophan, L-valine, L-tyrosine, L-cysteine, L-cystine disodium, L-hydroxyproline, and L-tyrosine disodium. These amino acids may be in the form of anhydrous, hydrated, solvated, or salt. Furthermore, the concentration of α-alanine in the medium of the present invention is preferably 60 to 300 mg / L, more preferably 80 to 150 mg / L. The β-alanine concentration is preferably 150 to 500 mg / L, more preferably 150 to 250 mg / L. The L-arginine-HCl concentration is preferably 400 to 800 mg / L. The L-aspartic acid concentration is preferably 250 to 1350 mg / L, more preferably 500 to 800 mg / L. The L-asparagine concentration is preferably 250 to 1350 mg / L, more preferably 500 to 800 mg / L. The L-glutamic acid concentration is preferably 500 to 1300 mg / L, more preferably 500 to 1000 mg / L. The L-glutamine concentration is preferably 500 to 1500 mg / L, more preferably 500 to 800 mg / L. The glycine concentration is preferably 200 to 650 mg / L, more preferably 200 to 400 mg / L. The L-histidine concentration is preferably 200 to 3500 mg / L, more preferably 700 to 1200 mg / L. The L-cystine concentration is preferably 20 to 130 mg / L, more preferably 50 to 80 mg / L. The L-isoleucine concentration is preferably 40 to 750 mg / L, more preferably 200 to 400 mg / L. The L-leucine concentration is preferably 60 to 250 mg / L, more preferably 100 to 200 mg / L.The concentration of L-lysine-HCl is preferably 400 to 1650 mg / L, more preferably 400 to 700 mg / L. The concentration of L-methionine is preferably 40 to 1000 mg / L, more preferably 300 to 600 mg / L. The concentration of L-phenylalanine is preferably 120 to 1000 mg / L, more preferably 300 to 600 mg / L. The concentration of L-proline is preferably 250 to 1700 mg / L, more preferably 200 to 400 mg / L. The concentration of DL-serine is preferably 250 to 950 mg / L, more preferably 400 to 700 mg / L. The concentration of L-threonine is preferably 100 to 350 mg / L, more preferably 100 to 200 mg / L. The concentration of L-tryptophan is preferably 50 to 100 mg / L. The L-valine concentration is preferably 80 to 500 mg / L, more preferably 150 to 300 mg / L. The L-tyrosine concentration is preferably 20 to 400 mg / L, more preferably 100 to 300 mg / L. The L-hydroxyproline concentration is preferably 200 to 400 mg / L.

[0022] The medium of the present invention may also contain an organic acid. Examples of such "organic acids" include malic acid, α-ketoglutaric acid, succinic acid, and fumaric acid. Such organic acids may be in the form of anhydrous, hydrated, solvated, or salt. In the medium of the present invention, the concentration of malic acid is preferably 200 to 550 mg / L, more preferably 200 to 400 mg / L. The concentration of α-ketoglutaric acid is preferably 100 to 200 mg / L. The concentration of succinic acid is preferably 15 to 50 mg / L, more preferably 15 to 30 mg / L. The concentration of fumaric acid is preferably 15 to 50 mg / L, more preferably 15 to 30 mg / L.

[0023] The medium of the present invention may also contain inorganic salts. Examples of such "inorganic salts" include NaHCO, MgSO7H0, NaHPO2H0, KCl, MgCl6H0, CaCl, CoCl6H0, and (NH)MoO. 24 Examples of inorganic salts include CuCl 4H2O, CuCl 2H2O, FeSO4 7H2O, MnCl2 4H2O, NaCl, ZnCl2, KH2PO4, NaH2PO4H2O, and MgCl2 4H2O. These inorganic salts may be in the form of anhydrous salts, hydrates, solvates, or salts. In the medium of the present invention, the NaHCO3 concentration is preferably 200 to 350 mg / L, more preferably 250 to 350 mg / L. The MgSO4 7H2O concentration is preferably 1000 to 2500 mg / L, more preferably 1000 to 2000 mg / L. The NaH2PO4 2H2O concentration is preferably 500 to 1500 mg / L, more preferably 700 to 1200 mg / L. The KCl concentration is preferably 1000 to 2000 mg / L, more preferably 1000 to 1600 mg / L. The MgCl26H2O concentration is preferably 600 to 2000 mg / L, more preferably 600 to 1000 mg / L. The CaCl2 concentration is preferably 400 to 850 mg / L, more preferably 400 to 700 mg / L. The CoCl26H2O concentration is preferably 0.02 to 0.5 mg / L, more preferably 0.02 to 0.05 mg / L. (NH4)6Mo7O 24 The concentration of 4H2O is preferably 0.01 to 0.02 mg / L. The concentration of CuCl22H2O is preferably 0.01 to 0.1 mg / L, more preferably 0.05 to 0.1 mg / L. The concentration of FeSO47H2O is preferably 0.1 to 0.3 mg / L. The concentration of MnCl24H2O is preferably 0.02 mg / L or less. The concentration of NaCl is preferably 1000 to 3500 mg / L. The concentration of ZnCl2 is preferably 0.01 to 0.02 mg / L.

[0024] The medium of the present invention may also contain vitamins. Examples of such "vitamins" include folic acid, biotin (vitamin H), riboflavin (vitamin B2), thiamine-HCl (vitamin B1-HCl), calcium pantothenate, pyridoxine-HCl, p-aminobenzoic acid, niacin (nicotinic acid), i-inositol, choline chloride, and vitamin B12. These vitamins may be in the form of anhydrous, hydrated, solvated, or salt. In addition, in the medium of the present invention, the folic acid concentration is preferably 0.05 to 0.16 mg / L. The biotin concentration is preferably 0.05 to 0.15 mg / L. The riboflavin concentration is preferably 0.05 to 0.16 mg / L. The thiamine-HCl concentration is preferably 0.05 to 0.16 mg / L. The calcium pantothenate concentration is preferably 0.05 to 0.16 mg / L. The pyridoxine-HCl concentration is preferably 0.15 to 0.3 mg / L. The p-aminobenzoic acid concentration is preferably 0.1 to 0.3 mg / L. The niacin concentration is preferably 0.1 to 0.2 mg / L. The i-inositol concentration is preferably 0.02 to 0.3 mg / L, more preferably 0.02 to 0.05 mg / L. The choline chloride concentration is preferably 1.5 to 12.0 mg / L, more preferably 5.0 to 12.0 mg / L. The vitamin B12 concentration is preferably 0.05 to 0.15 mg / L.

[0025] In addition to the above, the medium of the present invention may also contain, for example, protein extracts (fetuin, cytochrome C, inosine, bovine serum albumin Fr. V, lactalbumin hydrolysate, yeast extract (e.g., TC-yeastlate, Yeastolate), tryptose phosphate broth, etc.) and viscosity aids (e.g., polyvinylpyrrolidone such as polyvinylpyrrolidone K-90). In the medium of the present invention, the fetuin concentration is preferably 5 to 20 mg / L, more preferably 5 to 10 mg / L. The cytochrome C concentration is preferably 30 to 100 mg / L, more preferably 30 to 50 mg / L. The inosine concentration is preferably 50 to 200 mg / L, more preferably 50 to 100 mg / L. The bovine serum albumin Fr. V concentration is preferably 3,000 to 10,000 mg / L, more preferably 3,000 to 5,000 mg / L. The concentration of tryptose phosphate broth is preferably 1500 to 3000 mg / L, and the concentration of polyvinylpyrrolidone K-90 is preferably 100 to 500 mg / L, more preferably 150 to 300 mg / L.

[0026] The medium containing the sugars, amino acids, organic acids, inorganic salts, vitamins, protein extracts, and viscosity aids may be a known basal medium for insect culture or a mixed medium thereof. Examples of such "basal media" include MX, IPL-41 complete medium, MM, TC-100, Schneider's Drosophila culture medium, Grace's insect medium, MGM-450, MGM-464, Sf-900 II, Sf-900 III, and EX-CELL® 420. If trehalose is added to such a basal medium or a mixed medium thereof at the above-mentioned high concentration, it can be used as the medium of the present invention. However, MX containing trehalose at the above-mentioned high concentration is preferred from the viewpoints of better maintaining the cell condition and facilitating adjustment of the composition and amounts of the components contained therein.

[0027] Furthermore, the medium of the present invention may contain other substances in addition to the above-mentioned trehalose, other sugars, amino acids, organic acids, inorganic salts, vitamins, protein extracts, and viscosity enhancers. Examples of such "other substances" include serum (e.g., fetal bovine serum (FBS)), antibiotics (e.g., penicillin, streptomycin, amphotericin B), and glutathione. The "osmolality" of the medium of the present invention is preferably 200 to 500 mOsmol / kg, more preferably 300 to 400 mOsmol / kg. The "pH" of the medium of the present invention is preferably 6.0 to 7.0, more preferably 6.2 to 6.5. Those skilled in the art can adjust the pH to the appropriate level by adding an acidic solution such as hydrochloric acid or a basic solution such as sodium hydroxide to the medium, as appropriate. The medium of the present invention can be in the form of a liquid, semi-solid (e.g., a medium containing very thin agar (soft agar or almost liquid agar)), or solid, but is usually in the form of a liquid medium (culture solution).

[0028] <Method for producing cultured insect cells> The present invention relates to a method for producing cultured insect cells, which comprises a step of culturing cells collected from an insect in the medium of the present invention described above.

[0029] The "insects" targeted by the present invention are not particularly limited as long as they are arthropods classified in the class Insecta of the subphylum Hexapoda, and examples thereof include arthropods belonging to the order Hemiptera (Hemiptera), Hymenoptera (Hymenoptera), Coleoptera (Coleoptera, Coleoptera), Diptera (Diptera), and Lepidoptera (Lepidoptera). More specifically, examples of the order Hemiptera include arthropods belonging to the suborder Stomatopoda, such as whiteflies and aphids, and the suborder Hemiptera, such as stink bugs. Examples of the order Hymenoptera include arthropods belonging to the suborder Hymenoptera, such as honeybees, and the suborder Sawfly.

[0030] Furthermore, the cells collected from such insects and cultured in the medium of the present invention are not particularly limited, and examples thereof include cells constituting embryonic tissue, fat body tissue, reproductive tissue (testes, ovaries), digestive system tissue, nervous system tissue, muscular system tissue, and epidermal system tissue. When cultured, the cells may be in the form of cells (cell clumps, etc.) dispersed from tissue by disruption (homogenization, etc.) or enzyme treatment (trypsin treatment, etc.), or may be in the form of tissue itself or fragments thereof obtained by cutting or other treatment.

[0031] In the present invention, "culturing" refers to the maintenance and / or proliferation of cells, and includes not only the culture stage (primary culture) in which cells collected from an insect are grown to a confluent state, but also the subsequent subculture or maintenance culture. Such culturing is typically carried out by placing the cells together with the medium of the present invention in a cell culture vessel such as a cell culture flask, and then replacing or replenishing the medium as appropriate. The "culture temperature" can be adjusted appropriately depending on the type of insect and its cells being cultured, but is typically 20 to 35°C. The "culture period" is not particularly limited, but is at least 2 months or more (e.g., 3, 4, or 5 months), preferably 6 months or more (e.g., 7, 8, 9, 10, or 11 months), and more preferably 1 year or more (e.g., 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 years). The "cultured insect cells" obtained through such culturing may be primary cultured cells or established cell lines established from the cells. [Example]

[0032] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. In these examples, MIPC-50, MIPC-30, IPL-41 complete medium, MM, TC-100, Schneider's Drosophila culture medium, Grace's insect medium (with additives), MGM-450, MGM-464, MX, or a mixture of these media were used for primary culture of various insects. The compositions of these media are shown in Tables 1 to 6, broken down by component. Although not shown in these tables, fetal bovine serum (FBS) was further added to each medium to a final concentration of 10 to 20 v / v% during primary culture.

[0033] MIPC-50 and MIPC-30 are newly developed media prepared by the present inventors, based on the composition of MX, but modified so that the sugar is primarily trehalose. The difference between MIPC-50 and MIPC-30 is that the concentrations of amino acids, organic acids, vitamins, etc. are lower in the latter than in the former. Furthermore, as shown in Table 7, the osmotic pressure of MIPC-50 and MIPC-30 is not significantly different from that of other existing media. The osmotic pressure was measured using a freezing point depression osmometer (Osmometer OM802-D (VOGEL)). Table 7 shows the osmotic pressure when each medium contains 20 v / v% FBS.

[0034] Regarding other existing media, IPL-41 complete medium is prepared by adding tryptose phosphate broth to IPL-41 Insect Medium (1X) (ThermoFisher, Catalog No. 11405081) to a concentration of 2600 mg / L (see Weiss SA. et al., In Vitro, 1981, Vol. 17, pp. 495-502). For MM, see Mitsuhashi J. et al., Contrib Boyce Thompson Inst, 1964, Vol. 22, pp. 435-460. For TC-100, see ThermoFisher, Catalog No. 13055025. Schneider's Drosophila medium is described in Imogene Schneider, J Exp Zool, 1964, Vol. 156, No. 1, pp. 91-103; ThermoFisher, Catalog Number: 21720024; Technical Resources-Media Formulations 11720-Schneider's Drosophila Medium, [online], ThermoFisher Scientific, Internet.<URL:https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.124.html> For information on Grace's Insect Medium (with additives), see TDCGRACE, Nature, 1962, Vol. 195, pp. 788-789; ThermoFisher, Catalog Number: 11605094. For information on MGM-450, see Mitsuhashi J. et al., Appl Entomol Zool, 1988, Vol. 23, pp. 488-490. For information on MGM-464, see Mitsuhashi J., In Vitro Cellular & Developmental Biology-Animal, 2001, Vol. 37, pp. 330-337. For information on MX, see U.S. Patent No. 7,074,612.

[0035] [Table 1]

[0036] [Table 2]

[0037] [Table 3]

[0038] [Table 4]

[0039] [Table 5]

[0040] [Table 6]

[0041] [Table 7]

[0042] Example 1 Primary culture of European honeybees First, using tweezers under a microscope, eggs (48 to 96 hours after laying) were removed from the honeycomb of a European honeybee and placed in a petri dish. 70% ethanol was added and the mixture was left to stand for 5 to 10 minutes (all subsequent procedures were performed in a clean bench). The ethanol surrounding the eggs was removed using a pipette. Next, approximately 1 ml of sterile distilled water was added and then removed, a process repeated three times. Similarly, the eggs were rinsed three times with culture medium. Primary culture of European honeybees was then attempted using either Method A or Method B below.

[0043] (Method A) 1 ml of culture medium was added and transferred to a 1.5 ml tube. After centrifugation at 300 x g for 3 minutes, the supernatant was removed. Then, using a plastic pestle, the eggs were homogenized by rotating the pestle three times. 1 ml of culture medium was added and the mixture was slowly pipetted 20 times with a 1000 μl pipetter. After centrifugation at 100 x g for 2 minutes, the supernatant was removed. 500 μl to 1 ml of culture medium was added and the mixture was transferred to a culture flask (12.5 cm 2 ) and cultured in an incubator at 30°C. Approximately one-third of the culture medium was replaced with fresh medium every 10 to 14 days.

[0044] (Method B) One ml of culture medium was added, and the embryo was transferred to a new dish. The eggshell was removed using tweezers. The removed embryo was cut into 2-3 pieces using a thin knife. The embryo was transferred to a culture flask along with the culture medium, and additional culture medium was added as needed. The embryo was cultured in an incubator at 30°C. Approximately one-third of the culture medium was replaced with fresh medium every 10-14 days.

[0045] The culture media used for primary culture of European honeybees were MIPC-50, MGM-450, IPL-41 complete medium, MX, MGM-450, Schneider's Drosophila culture medium, and L-glutamine-supplemented MGM-450. MGM-450 and Schneider's Drosophila culture medium were prepared by mixing MGM-450 and Schneider's Drosophila culture medium in a 1:1 (volume ratio). L-glutamine-supplemented MGM-450 was prepared by mixing MGM-450 and 200 mM L-glutamine in a 10:1 (volume ratio). Each medium was supplemented with 10-20% (v / v) FBS during culture.

[0046] Example 2 Primary culture of Bemisia tabaci The eggs and ovaries of adult females of Bemisia tabaci were subjected to the following methods C and D, respectively, to attempt primary culture of Bemisia tabaci.

[0047] (Method C) Cabbage leaves (6-9 leaves, approximately 8 cm square) on which Bemisia tabaci eggs were laid were prepared. Adults were removed by suction, and the cabbage leaves were sprayed with 70% ethanol (all subsequent procedures were performed in a clean bench). 70% ethanol was prepared in a petri dish, and eggs were scraped off the cabbage leaves using a short brush. The leaves were then transferred to a 15 ml tube along with the ethanol and centrifuged at 300 x g for 3 minutes, after which the supernatant (ethanol) was removed (this process took approximately 15 minutes from the time of ethanol spraying). 1 ml of culture medium was added, and the mixture was transferred to a 1.5 ml tube. This process of centrifugation at 300 x g for 3 minutes and removal of the supernatant was repeated three times. The eggs were then homogenized using a plastic pestle by rotating it 5-8 times. 1 ml of culture medium was added, and the mixture was slowly pipetted 20 times using a 1000 μl pipetter. The homogenate was then passed through a 70 μm filter placed in a 15 ml tube. 1 ml of culture medium was added to the original 1.5 ml tube, washed, and passed through the filter. The tube was centrifuged at 100 × g for 2 minutes, the supernatant was removed, and 1 ml of culture medium was added. The tube was centrifuged at 100 × g for 2 minutes, and the supernatant was removed. 500 μl to 1 ml of culture medium was added, and the tube was placed in an organ culture dish (or culture flask, 24-well plate), wrapped in parafilm, and cultured in an incubator at 25°C. Approximately one-third of the culture medium was replaced with fresh medium every 10 to 14 days.

[0048] (Method D) Cabbage leaves (6–9 leaves, approximately 8 cm square) on which Bemisia tabaci eggs were laid were prepared. Adult insects were collected in a 1.5 ml tube by pumping. 70% ethanol was added to the tube, and after 1 minute and 30 seconds, the leaves were spread onto sterilized filter paper using a pipette. While the leaves were drying, the males and females were separated under a stereomicroscope. Culture medium was then prepared in a petri dish, and ovaries were extracted from the abdomen into the culture medium using two fine needles. Culture medium was prepared in a separate petri dish, and the ovaries were transferred into it using a pipette. The leaves were transferred to an organ culture dish (or 24-well plate), and 0–500 μl of culture medium was added as appropriate. The leaves were then wrapped in parafilm and cultured in an incubator at 25°C. Approximately one-third of the culture medium was replaced with fresh medium every 10–14 days.

[0049] The culture media used for primary culture of Bemisia tabaci were MIPC-50, MIPC-30, MGM-450, MGM-450, Schneider's Drosophila culture medium, and L-glutamine-supplemented MGM-450. Each medium was supplemented with 10-20% (v / v) FBS.

[0050] Example 3 Primary culture of pea aphids Adult female pea aphids were collected in a petri dish. They were sterilized with 70% ethanol for 5-10 minutes (subsequent procedures were performed in a clean bench). They were air-dried on a sterilized paper towel, and embryos were removed from their abdomens using tweezers in culture medium (approximately 1 ml) placed in a petri dish. The culture medium was transferred to a 1.5 ml tube and gently spun down to remove the supernatant. 1 ml of culture medium was added, spun down again to remove the supernatant, and the embryos were homogenized using a plastic pestle by rotating the pestle five times. 1 ml of culture medium was added, and the mixture was slowly pipetted 20 times with a 1000 μl pipetter. The homogenized liquid was then passed through a 70 μm filter placed in a 15 ml tube. 1 ml of culture medium was then added to the original 1.5 ml tube, washed, and passed through the filter. The mixture was centrifuged at 100×g for 2 minutes, the supernatant was removed, 1 ml of culture medium was added, the mixture was centrifuged at 100×g for 2 minutes, the supernatant was removed, 1 ml of culture medium was added, and the mixture was gently pipetted and transferred to a culture flask (12.5 cm 2 Alternatively, 500 μl to 1 ml of culture medium was added, placed in an organ culture dish, wrapped in parafilm, and cultured in an incubator at 20°C. Approximately one-third of the culture medium was replaced with fresh medium every 10 to 14 days.

[0051] The culture media used for primary culture of the pea aphid were MIPC-50, MIPC-30, IPL-41 complete medium, MM, MGM-450, MGM-464, and MX. Each medium was supplemented with 10-20% (v / v) FBS.

[0052] Example 4 Primary culture of German-winged green bugs German-winged green stink bug eggs (stage where the compound eyes are visible through the red light) were collected. They were sterilized with 70% ethanol for 5-10 minutes (all subsequent procedures were carried out in a clean bench). They were air-dried on a sterilized paper towel, and the eggshells were removed using tweezers in a petri dish containing culture medium (approximately 1 ml). The eggs were transferred to a 1.5 ml tube along with the culture medium and slowly pipetted 20 times with a 1000 μl pipetter. The mixture was centrifuged at 100 x g for 2 minutes, and the supernatant was carefully removed. The fat layer on top was particularly removed. The embryos were homogenized using a plastic pestle by rotating the pestle five times. Add 1 ml of culture medium, pipette gently, and place in a culture flask (12.5 cm 2 ) and 4 ml of culture medium was added (total of 5 ml). Alternatively, 500 μl of culture medium was added, gently pipetted, and transferred to an organ culture dish. After 7 days, 500 μl to 1 ml of culture medium was added (total of 1 to 1.5 ml). Then, the cells were cultured in an incubator at 25°C. Approximately one-third of the culture medium was replaced with fresh medium every 10 to 14 days.

[0053] The culture media used for primary culture of the German-winged green bug were MIPC-50 and MX. Note that 10-20 v / v% FBS was added to each medium during culture.

[0054] The results of primary culture using the above various media are shown below. Table 8 also shows the longest survival period of primary cultured cells derived from each insect in each culture medium. The concentration of FBS added to the medium used for culture is shown in parentheses for each item in Table 8. For the European honeybee and Bemisia tabaci, the culture methods (Methods A to D above) used for culture are also shown in parentheses.

[0055] Example 1 When MX was used in primary culture of honeybees, the tissue adhered to the container after the start of culture and cell migration was observed. The cells then died after about three months. When MGM-450 was used, no cell proliferation was observed and the cells died after about two months. When MGM-450 and Schneider's Drosophila culture medium were used, the tissue adhered to the container after the start of culture and cell migration was observed. The cells were then maintained for about six months, but were not subcultured. When L-glutamine-supplemented MGM-450 was used, a small amount of cell migration was observed after the start of culture, but there was no subsequent proliferation and the cells died after about five months.

[0056] On the other hand, when MIPC-50 was used, a large number of cells adhered to the flask after the start of culture, and proliferation was observed. After approximately two months, the cells proliferated until they reached confluence. When subculture was attempted, the cells were maintained for three generations, but after that, the adhered cells began to float, the cell number decreased, and the cells could no longer be maintained. As a result, viable cells were confirmed for approximately one year and six months after the start of culture when culturing cells derived from European honeybees using MIPC-50.

[0057] Example 2 When primary cultures of Bemisia tabaci were performed using MGM-450, MGM-450 and Schneider's Drosophila culture medium, or MGM-450 supplemented with L-glutamine, almost no cells adhered to the culture vessel after the start of culture, and floating cells died within about one month.

[0058] On the other hand, when MIPC-50 or MIPC-30 was used, cell clumps were formed in the early stages of culture. After that, the cells proliferated slightly, but did not reach a density sufficient for subculture. However, they were able to survive for more than 7 months after the start of culture.

[0059] Example 3 When primary cultures of pea aphids were cultured on IPL-41 complete medium, few cells adhered to the flask after the start of culture, and many spherical cells were floating. After about a month and a half, viable cells disappeared. When MM was used, many cultured tissues and cells swelled, with only a few attached cells. After about 20 days, viable cells were no longer observed. When MGM-450 was used, many cells adhered to the flask, and after a while, cell elongation was observed. However, viable cells disappeared after about two months. When MGM-464 was used, few cells and tissues adhered to the flask, and some floating cells survived for about three months. When MX was used, many cells adhered to the flask. The floating tissues swelled slightly. After that, the attached cells floated, but a very few viable cells remained. However, viable cells disappeared after about three months.

[0060] On the other hand, when MIPC-50 was used, many cells adhered together with the tissue. Afterwards, vesicles were formed, but these disappeared after about a month. The cells then survived for about four months. When MIPC-30 was used, many cells adhered to the flask and spread out after a while. There were floating spherical cell clusters, which survived for about five and a half months.

[0061] Example 4 When MX was used in primary culture of German-winged green stink bugs, cell migration was observed immediately after the start of culture, but the number of surviving cells subsequently decreased. The cells survived for about three months.

[0062] On the other hand, when MIPC-50 was used, there were few adherent cells immediately after the start of culture, but vesicle-like cells were present. Subsequently, more cells began to adhere, and after three months, various cell types, including adherent epidermis-like cells and spindle-shaped cells, began to proliferate. After nine months, the cell numbers had increased to the point where they could be passaged. Furthermore, when subculture was attempted, the rate of proliferation was slow, but the cells are still maintained after one year and nine months. Furthermore, when a separate sample was cultured on MIPC-50, cell migration was observed two weeks after the start of culture, and some adhered to form sheets, spindle-shaped cells, and vesicle-like cells were observed. Subculture of some of the cells was then performed, and they are still maintained, with viable cells present, approximately two years and four months later (however, at this time, there has been no proliferation sufficient to permit further passage).

[0063] [Table 8] [Industrial Applicability]

[0064] As described above, the present invention makes it possible to culture various insect cells. Therefore, the present invention is useful in many fields in which insect cells can be used, such as gene function analysis, protein expression, insect microbiology research, and drug development. For example, in the field of pesticide development, the use of the various insect cell culture systems according to the present invention makes it possible to easily evaluate the efficacy of pesticides.

Claims

1. A medium for culturing insect cells, comprising 10 to 20 g / L of trehalose.

2. The medium of claim 1 , wherein the culture is a primary culture.

3. A method for producing cultured insect cells, comprising a step of culturing cells collected from an insect in the medium according to claim 1.

4. The method according to claim 3, wherein the cultured insect cells are primary cultured cells.

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