Method for stabilizing extracellular vesicles

Abstract

To provide stabilization of an extracellular vesicle.SOLUTION: The present invention provides, for example, a method of stabilizing an extracellular vesicle, including mixing an extracellular vesicle-containing sample with a saccharide and a chelating agent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for stabilizing extracellular vesicles and the like.

Background Art

[0002] Extracellular vesicles are minute vesicles having a membrane structure, which are secreted from various types of cells and present in body fluids such as blood. Extracellular vesicles secreted extracellularly include exosomes, ectosomes, and apoptotic blebs. Since extracellular vesicles contain various substances responsible for functions such as intercellular information transmission, they have been analyzed for purposes such as diagnosis and drug discovery. Therefore, development of a method for treating extracellular vesicles useful for such analysis has been demanded. For example, Patent Document 1 discloses that (1) although the yield of extracellular vesicles can be improved in the presence of a low concentration of EDTA (chelating agent) of about 3.0 mg / mL (about 10 m M) or less, the yield of extracellular vesicles cannot be improved in the presence of a higher concentration of EDTA (Examples 1, FIGS. 1A to 1C), (2) extracellular vesicles are stored at a predetermined temperature and time in the presence of a low concentration of EDTA of about 2.25 mg / mL (about 7.7 m M) (Examples 2, FIGS. 2A and 2B, FIGS. 3A to 3F), and (3) extracellular vesicles are frozen and thawed in the presence of a low concentration of EDTA of about 2.25 mg / mL (about 7.7 μM) (Example 3, FIGS. 4A and 4B).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] If extracellular vesicles can be stabilized, the preservation of extracellular vesicles can be improved, which is useful for applications in fields such as diagnosis and drug discovery. Therefore, an object of the present invention is to develop a method for stabilizing extracellular vesicles.

Means for Solving the Problems

[0005] As a result of intensive studies, the present inventors have found that extracellular vesicles in a sample containing extracellular vesicles can be stabilized by mixing the sample with a predetermined component such as sugar, and have thus completed the present invention.

[0006] That is, the present invention is as follows. 〔1〕A method for stabilizing extracellular vesicles, comprising mixing a sample containing extracellular vesicles with a sugar and a chelating agent. 〔2〕The method according to 〔1〕, wherein the extracellular vesicles are exosomes. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the sample containing extracellular vesicles is a body fluid or a culture supernatant. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein the sample containing extracellular vesicles is a blood sample. 〔5〕The method according to any one of 〔1〕 to 〔4〕, wherein the concentration of the sugar in the mixing is 2.5 to 100 mg / mL. 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the concentration of the chelating agent in the mixing is 1 to 200 mM. 〔7〕A method for stabilizing extracellular vesicles, comprising: (1) mixing a sample containing extracellular vesicles with a sugar; and (2) freezing the mixture of the sample containing extracellular vesicles and the sugar. 〔8〕The method according to 〔7〕, wherein the freezing is freeze-drying. 〔9〕A method for stabilizing extracellular vesicles, comprising: (1) mixing a sample containing extracellular vesicles with a chelating agent; and (2) freezing the mixture of the sample containing extracellular vesicles and the chelating agent. 〔10〕The method according to 〔9〕, wherein the freezing is freeze-drying. 〔11〕An extracellular vesicle stabilizing reagent containing a sugar and a chelating agent. 〔12〕A cryopreservation stabilizing reagent for extracellular vesicles containing sugar or a chelating agent.

Advantages of the Invention

[0007] By mixing a sample containing extracellular vesicles with sugar, the extracellular vesicles in the sample containing extracellular vesicles can be stabilized. Therefore, the present invention is useful, for example, for the preservation of extracellular vesicles.

Brief Description of the Drawings

[0008]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0009] 1. Method for stabilizing extracellular vesicles The present invention provides a method for stabilizing extracellular vesicles, which includes mixing a sample containing extracellular vesicles with a sugar.

[0010] The present invention also provides a method for stabilizing extracellular vesicles, which includes mixing a sample containing extracellular vesicles with a chelating agent.

[0011] The present invention also provides a method for stabilizing extracellular vesicles, which includes mixing a sample containing extracellular vesicles with a sugar and a chelating agent.

[0012] Extracellular vesicles are tiny vesicles with a membrane structure secreted from various types of cells. Examples of extracellular vesicles include exosomes, ectosomes, and apoptotic bodies. Preferably, the extracellular vesicles are exosomes. Extracellular vesicles can also be defined by their size. The size of extracellular vesicles is, for example, 30 to 1000 nm, preferably 50 to 300 nm, more preferably 80 to 200 nm. The size of extracellular vesicles can be measured by, for example, methods based on the Brownian motion of extracellular vesicles, light scattering methods, and electrical resistance methods. Preferably, the size of extracellular vesicles is measured by NanoSight LM10 (manufactured by Malvern Instruments). When using NanoSight LM10, as measurement conditions, a measurement time of 30 seconds, a repetition number of 3 times, and a detection threshold of 15 can be adopted. Extracellular vesicles can further be defined by extracellular vesicle markers. Examples of extracellular vesicle markers include CD9, carcinoembryonic antigen (CEA), CD81, CD63, heat shock protein (HSP) 70, HSP90, major histocompatibility complex (MHC) I, tumor susceptibility gene (TSG) 101, lysosome-associated membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I, Flotillin-I, Rab protein, EpCAM.

[0013] An extracellular vesicle-containing sample is any sample containing extracellular vesicles. Preferably, the extracellular vesicle-containing sample is a biological liquid sample. The extracellular vesicle-containing sample may be subjected to other treatments before being used in the method of the present invention. Examples of such treatments include centrifugation, extraction, filtration, precipitation, heating, refrigeration, and stirring.

[0014] In one embodiment, the extracellular vesicle-containing sample is a culture supernatant. The culture supernatant may be a cell culture supernatant or a tissue culture supernatant. Examples of the organism from which the cells or tissue to be cultured are derived include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; domestic animals such as cows, pigs, and goats; and draft animals such as horses and sheep), birds (e.g., chickens), insects, microorganisms (e.g., bacteria), plants, and fish. Preferably, the organism is a mammal such as a human.

[0015] In another embodiment, the extracellular vesicle-containing sample is a body fluid. The body fluid is a body fluid derived from an organism as described above. Examples of the body fluid include blood samples (e.g., whole blood, serum, and plasma), lymph, tissue fluid, cerebrospinal fluid, ascites, saliva, pancreatic juice, bile, sweat, semen, urine, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Preferably, the body fluid is blood.

[0016] In yet another embodiment, the extracellular vesicle-containing sample is milk or fruit juice.

[0017] The sugar is a monosaccharide (e.g., aldose, ketose) or a polysaccharide in which two or more monosaccharides are linked by a glycosidic bond. In the present invention, the sugar also includes derivatives and sugar alcohols. A sugar derivative refers to a compound in which a hydrogen atom, a hydroxyl group, or a carbonyl group in the sugar is substituted with a substituent. A sugar alcohol refers to a compound in which the carbonyl group in the sugar is reduced.

[0018] More specifically, examples of monosaccharides include trioses (e.g., glyceraldehyde, dihydroxyacetone), tetroses (e.g., erythrose, threose, erythrulose), pentoses (e.g., xylose, ribose, arabinose, lyxose, ribulose, xylulose, apiose), hexoses (e.g., glucose, fructose, galactose, mannose, allose, altrose, gulose, idose, talose, psicose, sorbose, tagatose), and heptoses (e.g., sedoheptulose, colios). Examples of aldoses include xylose, glucose, galactose, mannose, glyceraldehyde, erythrose, threose, ribose, arabinose, lyxose, allose, altrose, gulose, idose, and talose. Examples of ketoses include fructose, dihydroxyacetone, erythrulose, xylulose, ribulose, psicose, sorbose, tagatose, sedoheptulose, and colios.

[0019] Examples of the polysaccharide include polysaccharides in which two or more of the above-described monosaccharides are linked. Preferably, the polysaccharide is a polysaccharide in which two or more monosaccharide units selected from the group consisting of glucose, fructose, galactose, mannose, and xylose are glycosidically bonded (e.g., a polysaccharide in which one or more glucoses and one or more fructoses are glycosidically bonded). Such a polysaccharide may be a linear polysaccharide or a cyclic polysaccharide. The linear polysaccharide may be a linear oligosaccharide or a linear high molecular polysaccharide. When the polysaccharide is a linear oligosaccharide, the total number of monosaccharide units in the polysaccharide may be, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 6, and even more preferably 2 to 4. For example, when the polysaccharide is a polysaccharide in which one or more glucoses and one or more fructoses are glycosidically bonded, the number of glucoses and fructoses in the polysaccharide may each independently be, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2. Examples of the linear oligosaccharide include disaccharides. Examples of the disaccharide include sucrose, lactose, and trehalose. Particularly preferably, the disaccharide is sucrose in which one glucose and one fructose are glycosidically bonded. Examples of the linear high molecular polysaccharide include cellulose, amylose, amylopectin, glucomannan, pullulan, galactomannan, inulin, glycogen, chitin, chitosan, glucuronoxylan, arabinoxylan, agarose, carrageenan, pectin, pectic acid, alginic acid, fucoidan, chondroitin sulfate, and hyaluronan. When the polysaccharide is a cyclic polysaccharide, the total number of monosaccharide units in the polysaccharide may be, for example, 5 to 100, preferably 5 to 20, more preferably 5 to 10, and even more preferably 6 to 8. Examples of the cyclic polysaccharide include cyclodextrin (α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin).

[0020] Examples of the substituent in the sugar derivative include, for example, a hydrogen atom, a hydroxyl group, C 1~6 alkyl group, C 1~6 alkenyl group, C 1~6 alkynyl group, C1~6 Alkyloxy(alkoxy) group, C 6~14 Aromatic hydrocarbon group, C 1~6 Examples thereof include an alkyl-carbonyl (acyl) group, a carboxy group, a nitro group, an amino group, and a cyano group.

[0021] C 1~6 The alkyl group is an alkyl having 1 to 6 carbon atoms, and may be linear, branched, or cyclic, but linear or branched alkyl is preferred. C 1~6 Examples of the alkyl group include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl. C 1~6 As the alkyl group, C 1~4 An alkyl group is preferred, and C 1~3 An alkyl group is more preferred. C 1~6 The alkenyl group is an alkenyl group having 1 to 6 carbon atoms, and may be linear, branched, or cyclic, but linear or branched alkenyl is preferred. C 1~6 Examples of the alkenyl group include ethenyl (vinyl), propenyl, butenyl, pentenyl, and hexenyl. C 1~6 As the alkenyl group, C 1~4 An alkenyl group is preferred, and C 1~3 An alkenyl group is more preferred.

[0022] C 1~6 The alkynyl group is an alkynyl group having 1 to 6 carbon atoms, and may be linear, branched, or cyclic, but linear or branched alkynyl is preferred. C 1~6 Examples of the alkynyl group include ethynyl, propynyl, butynyl, pentynyl, and hexynyl. C 1~6 As the alkynyl group, C 1~4 An alkynyl group is preferred, and C 1~3 An alkynyl group is more preferred. C 1~6 The alkyloxy group is an alkyloxy group having 1 to 6 carbon atoms. C 1~6 Examples of the alkyloxy group include methyloxy, ethyloxy, propyloxy, iso-propyloxy, butyloxy, iso-butyloxy, sec-butyloxy, tert-butyloxy, pentyloxy, isopentyloxy, neopentyloxy, 1-ethylpropyloxy, hexyloxy, isohexyloxy, 1,1-dimethylbutyloxy, 2,2-dimethylbutyloxy, 3,3-dimethylbutyloxy, 2-ethylbutyloxy. C 1~6 As the alkyloxy group, C 1~4 an alkyloxy group is preferred, C 1~3 and an alkyloxy group is more preferred.

[0023] C 6~14 Examples of the aromatic hydrocarbon group include phenyl, naphthyl, anthracenyl. C 6~14 As the aromatic hydrocarbon group, phenyl and naphthyl are preferred, and phenyl is more preferred.

[0024] C 1~6 The alkyl-carbonyl (acyl) group is a carbonyl group having an alkyl group as described above. C 1~6 C 1~6 Examples of the alkyl-carbonyl group include methylcarbonyl (acetyl), ethylcarbonyl, propylcarbonyl, iso-propylcarbonyl, butylcarbonyl, iso-butylcarbonyl, sec-butylcarbonyl, tert-butylcarbonyl, pentylcarbonyl, isopentylcarbonyl, neopentylcarbonyl, 1-ethylpropylcarbonyl, hexylcarbonyl, isohexylcarbonyl, 1,1-dimethylbutylcarbonyl, 2,2-dimethylbutylcarbonyl, 3,3-dimethylbutylcarbonyl, 2-ethylbutylcarbonyl. C 1~6 As the alkylcarbonyl group, C 1~4 an alkylcarbonyl group is preferred, C 1~3 and an alkylcarbonyl group is more preferred.

[0025] These substituents may be further substituted with another substituent. Examples of the other substituent include a hydroxyl group, a carboxy group, a nitro group, an amino group, and a cyano group.

[0026] Examples of the derivative of the sugar substituted with a substituent include derivatives of cyclic polysaccharides (e.g., cyclodextrins such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin) and chain-like high molecular weight polysaccharides (e.g., cellulose). Examples of the derivative of the cyclic polysaccharide include a cyclic polysaccharide substituted with an optionally substituted alkyl group (e.g., methyl β-cyclodextrin, hydroxypropyl β-cyclodextrin). Examples of the derivative of the chain-like high molecular weight polysaccharide include a cellulose derivative.

[0027] The cellulose derivative is a cellulose derivative in which a hydrogen atom of at least one hydroxyl group of cellulose is substituted with a hydrophilic group. Examples of the hydrophilic group in the cellulose derivative include carboxyalkyl (e.g., carboxy C 1~6 alkyl), hydroxyalkyl (e.g., hydroxy C 1~6 alkyl). The hydrophilic group in the cellulose derivative is preferably carboxyalkyl or hydroxyalkyl. Examples of carboxyalkyl include carboxymethyl, carboxyethyl (1-carboxyethyl, 2-carboxyethyl), carboxypropyl (1-carboxypropyl, 2-carboxypropyl, 3-carboxypropyl), carboxyisopropyl (1-carboxy-2-methylethyl, 2-carboxy-2-methylethyl), carboxybutyl (1-carboxybutyl, 2-carboxybutyl, 3-carboxybutyl, 4-carboxybutyl), carboxyt-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl, 3-carboxypentyl, 4-carboxypentyl, 5-carboxypentyl), carboxyhexyl (1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl, 5-carboxyhexyl, 6-carboxyhexyl). Examples of hydroxyalkyl include hydroxymethyl, hydroxyethyl (1-hydroxyethyl, 2-hydroxyethyl), hydroxypropyl (1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl), hydroxyisopropyl (1-hydroxy-2-methylethyl, 2-hydroxy-2-methylethyl), hydroxybutyl (1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl), hydroxyt-butyl, hydroxypentyl (1-hydroxypentyl, 2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl, 5-hydroxypentyl), hydroxyhexyl (1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl, 5-hydroxyhexyl, 6-hydroxyhexyl). Specific examples of cellulose derivatives include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose. Cellulose derivatives include nanocellulose derivatives. Nanocellulose derivatives are derivatives of nanocellulose, which will be described later.

[0028] Nanocellulose is fibrous cellulose having a fiber width on the order of nanometers. The fiber width of nanocellulose is, for example, 500 nm or less, preferably 200 nm or less, more preferably 100 nm or less, even more preferably 50 nm or less, still even more preferably 10 nm or less, and particularly even more preferably 5 nm or less.

[0029] Cellulose derivatives also include their salts. Examples of salts include salts of metals (e.g., monovalent metals such as lithium, sodium, potassium, rubidium, and cesium, and divalent metals such as calcium, magnesium, and zinc), as well as salts of inorganic bases (e.g., ammonia).

[0030] Examples of sugar alcohols include monomers in which the carbonyl group in the above-mentioned monosaccharides is reduced, polymers in which these monomers are bonded to each other, for example, by a glycosidic bond, and polymers in which these monomers and monosaccharides are bonded as constituent units, for example, by a glycosidic bond. Examples of the above monomers include tritols (e.g., glycerin), tetritols (e.g., erythritol, threitol), pentitols (e.g., xylitol, arabinitol, ribitol), hexitols (e.g., sorbitol, mannitol, iditol, galactitol), heptitols (e.g., volemitol, perseitol), and octitols (e.g., erythrogalactooctitol). Examples of sugar alcohols include monomers such as xylitol, sorbitol, mannitol, galactitol, fucitol, volemitol, arabinitol, glycerin, iditol, erythritol, threitol, ribitol, and dimers such as lactitol and maltitol.

[0031] In the present invention, a mixture of two or more (e.g., two, three, four, five) sugars may be mixed with the extracellular vesicle-containing sample. In the present invention, before mixing the extracellular vesicle-containing sample with the sugar, it may include adding the sugar to the extracellular vesicle-containing sample. The sugar may be added simultaneously or separately from the chelating agent.

[0032] When mixing an extracellular vesicle-containing sample with a sugar, the concentration of the sugar in the mixture is not particularly limited as long as it can stabilize the extracellular vesicles. Such a concentration varies depending on factors such as the type of sugar. For example, it may be 2.5 mg / mL or more, preferably 3.0 mg / mL or more, more preferably 3.5 mg / mL or more, even more preferably 4.0 mg / mL or more, still even more preferably 4.5 mg / mL or more, particularly preferably 5.0 mg / mL or more, 6.0 mg / mL or more, 8.0 mg / mL or more, or 10.0 mg / mL or more. Such a concentration also varies depending on factors such as the type of sugar. For example, it may be 600 mg / mL or less, 400 mg / mL or less, 200 mg / mL or less, or 100 mg / mL or less, preferably 95 mg / mL or less, more preferably 90 mg / mL or less, even more preferably 85 mg / mL or less, still even more preferably 80 mg / mL or less, particularly preferably 75 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, or 50 mg / mL or less. More specifically, the concentration of the sugar varies depending on factors such as the type of sugar. For example, it may be 2.5 to 800 mg / mL, 2.5 to 600 mg / mL, 2.5 to 400 mg / mL, 2.5 to 200 mg / mL, or 2.5 to 100 mg / mL, preferably 3.0 to 95 mg / mL, more preferably 3.5 to 90 mg / mL, even more preferably 4.0 to 85 mg / mL, still even more preferably 4.5 to 80 mg / mL, particularly preferably 5.0 to 75 mg / mL, 6.0 to 70 mg / mL, 8.0 to 60 mg / mL, or 10.0 to 50 mg / mL.

[0033] A chelating agent is a compound having a coordination moiety capable of forming a coordination bond with a metal ion or a salt thereof. The number of coordination moieties is preferably 2 or more, more preferably 3 or more (e.g., 3 or 6). Examples of the coordinating atom as the coordination moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordinating atom is preferably an oxygen atom or a phosphorus atom, more preferably an oxygen atom. Examples of the coordinating group as the coordination moiety include a group having the above-mentioned coordinating atom. The coordinating group is preferably a carboxylic acid group or a phosphoric acid group, more preferably a carboxylic acid group.

[0034] Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), glycol ether diamine tetraacetic acid (EGTA), hydroxyethyl ethylenediamine triacetic acid (HEDTA), hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediamine tetra(methylenephosphonic acid) (EDTMP), and salts thereof. Examples of the salts include metal salts (e.g., monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt), inorganic salts (e.g., halide salts such as fluoride, chloride, bromide, and iodide, and ammonium salts), organic salts (e.g., ammonium salts substituted with an alkyl group), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid). In the present invention, a mixture of two or more (e.g., 2, 3, 4, 5) chelating agents may be mixed with the extracellular vesicle-containing sample. In the present invention, it may include adding a chelating agent to the extracellular vesicle-containing sample before mixing the extracellular vesicle-containing sample with the chelating agent.

[0035] When mixing an extracellular vesicle-containing sample with a chelating agent, the concentration of the chelating agent in the mixing is not particularly limited as long as it can stabilize the extracellular vesicles. Such a concentration varies depending on factors such as the type of chelating agent, but for example, it may be 1 mM or more, preferably 5 mM or more, more preferably 10 mM or more, even more preferably 15 mM or more, still even more preferably 20 mM or more, particularly preferably 30 mM or more, 40 mM or more, or 50 mM or more. Such a concentration also varies depending on factors such as the type of chelating agent, but for example, it may be 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, even more preferably 160 mM or less, still even more preferably 150 mM or less, particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less. More specifically, the concentration of the chelating agent may be, for example, 1 mM to 200 mM, preferably 5 to 180 mM, more preferably 10 to 170 mM, even more preferably 15 to 160 mM, still even more preferably 20 to 150 mM, particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

[0036] The extracellular vesicle-containing sample may be mixed with both sugar and a chelating agent.

[0037] When both sugar and a chelating agent are used in the present invention, the concentrations of the sugar and the chelating agent used in the mixing can also be defined by their ratio. For example, the concentration of the chelating agent per 10 mg / mL of sugar in the mixing varies depending on factors such as the types of the sugar and the chelating agent, but may be, for example, 1 mM or more, preferably 5 mM or more, preferably 10 mM or more, more preferably 15 mM or more, even more preferably 20 mM or more, particularly preferably 30 mM or more, 40 mM or more, or 50 mM or more. Such concentrations also vary depending on factors such as the types of the sugar and the chelating agent, but may be, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, more preferably 160 mM or less, even more preferably 150 mM or less, particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less. More specifically, the concentration of the chelating agent may be, for example, 1 mM to 200 mM, preferably 5 to 180 mM, more preferably 10 to 170 mM, more preferably 15 to 160 mM, even more preferably 20 to 150 mM, particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

[0038] The mixing can be carried out in any manner. For example, when both sugar and a chelating agent are used in the present invention, the mixing can be carried out simultaneously or separately. More specifically, the extracellular vesicle-containing sample may be (1) mixed with the sugar and the chelating agent simultaneously, (2) mixed with the sugar first and then with the chelating agent, or (3) mixed with the chelating agent first and then with the sugar. From the viewpoint of simplicity of operation, etc., the extracellular vesicle-containing sample may preferably be mixed with the sugar and the chelating agent simultaneously.

[0039] The mixing temperature is, for example, 4 to 37 °C, and may preferably be 15 to 30 °C. The mixing time is not particularly limited as long as the extracellular vesicles can be stabilized, and can be set as appropriate. The extracellular vesicle-containing sample may be left standing after mixing with a predetermined component such as sugar.

[0040] Whether extracellular vesicles are stabilized by mixing with a predetermined component such as sugar can be determined, for example, by comparing an index value measured after mixing extracellular vesicles with a predetermined component such as sugar with a control value measured for extracellular vesicles in the absence of a predetermined component such as sugar (where conditions other than the presence or absence of a predetermined component such as sugar are the same), and evaluating whether the index value is superior to the control value. For example, when evaluating the stabilization of a lyophilized product, a freeze dryer (manufactured by Tokyo Rika Kikai Co., Ltd.) can be used to perform lyophilization at -28°C for 2 hours, -10°C for 4 hours, and then at 20°C according to a program, and the index values of the lyophilized products in the presence or absence of a predetermined component such as sugar can be compared. As such an index value, for example, the amount of extracellular vesicle markers or the measured value of the number of particles corresponding to extracellular vesicles can be used.

[0041] The measurement of the amount of extracellular vesicle markers can be performed by any method known in the art. When the extracellular vesicle marker is a protein, examples of the measurement method include immunoassay and mass spectrometry. Examples of immunoassay include direct competition method, indirect competition method, and sandwich method. Further, such immunoassays include chemiluminescent immunoassay (CLIA) [e.g., chemiluminescent enzyme immunoassay (CLEIA)], turbidimetric immunoassay (TIA), enzyme immunoassay (EIA) (e.g., direct competition ELISA, indirect competition ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), and immunochromatography method, Western blotting, immunostaining, fluorescence activated cell sorting (FACS). When multiple components are detected, proteome analysis may be performed. When the extracellular vesicle marker is a nucleic acid, examples of the measurement method include hybridization method using a probe, gene amplification method using primers (e.g., 2, 3, or 4 primers), and mass spectrometry. When the extracellular vesicle marker is a component other than proteins and nucleic acids, examples of the measurement method include immunoassay and mass spectrometry. When multiple components are measured, metabolome analysis may be performed.

[0042] The measurement of the number of particles corresponding to extracellular vesicles can be performed by devices such as particle analyzers, electron microscopes, and flow cytometers. Preferably, such measurement of the number of particles can be performed using NanoSight LM10 (manufactured by Malvern Instruments). When using NanoSight LM10, as measurement conditions, a measurement time of 30 seconds, a repetition number of 3 times, and a detection threshold of 15 can be adopted.

[0043] The method of the present invention may further include freezing the mixture.

[0044] That is, the present invention further provides a method for stabilizing extracellular vesicles, including the following: (1) Mixing a sample containing extracellular vesicles with sugar; and (2) Freezing the mixture of the sample containing extracellular vesicles and sugar.

[0045] The present invention further provides a method for stabilizing extracellular vesicles, including the following: (1) Mixing a sample containing extracellular vesicles with a chelating agent; and (2) Freezing the mixture of the sample containing extracellular vesicles and the chelating agent.

[0046] The present invention further provides a method for stabilizing extracellular vesicles, including the following: (1) Mixing a sample containing extracellular vesicles with sugar and a chelating agent; and (2) Freezing the mixture of the sample containing extracellular vesicles and sugar and the chelating agent.

[0047] The freezing may be either freeze-drying or freezing of the solution (non-dry freezing), but freeze-drying is preferred when storing extracellular vesicles over a longer period.

[0048] Examples of the types of sugars and chelating agents and their concentrations in the mixture are as described above.

[0049] 2. Extracellular vesicle stabilizing reagent The present invention also provides an extracellular vesicle stabilizing reagent. The reagent of the present invention contains a sugar or a chelating agent. Preferably, the reagent of the present invention may contain a sugar and a chelating agent. The sugar and the chelating agent are the same as those described above. The reagent of the present invention may contain other components (e.g., other components useful for stabilizing extracellular vesicles) in addition to the sugar and the chelating agent.

[0050] In one embodiment, the reagent of the present invention is an extracellular vesicle stabilizing composition containing a sugar. In another embodiment, the reagent of the present invention is an extracellular vesicle stabilizing composition containing a chelating agent. In yet another embodiment, the composition of the present invention is an extracellular vesicle stabilizing composition containing both a sugar and a chelating agent.

[0051] To stabilize extracellular vesicles, the reagent of the present invention can be used by mixing it with a sample containing extracellular vesicles. To stably preserve extracellular vesicles, the mixture of the sample containing extracellular vesicles and the reagent of the present invention may be frozen. In this case, the reagent of the present invention can be used as a cryopreservation stabilizing reagent for extracellular vesicles. Freezing may be either freeze-drying or freezing of a solution (non-dry freezing), but freeze-drying is preferred when preserving extracellular vesicles over a longer period.

[0052] The concentration of sugar in the composition is not particularly limited as long as it can stabilize extracellular vesicles after being mixed with the extracellular vesicle-containing sample. Such a concentration varies depending on factors such as the type of sugar. For example, it may be 2.7 mg / mL or more, preferably 3.0 mg / mL or more, more preferably 3.5 mg / mL or more, even more preferably 4.0 mg / mL or more, still even more preferably 4.5 mg / mL or more, particularly preferably 5.0 mg / mL or more, 6.0 mg / mL or more, 8.0 mg / mL or more, or 10.0 mg / mL or more. Such a concentration also varies depending on factors such as the type of sugar. For example, it may be 600 mg / mL or less, 400 mg / mL or less, 200 mg / mL or less, or 100 mg / mL or less, preferably 95 mg / mL or less, more preferably 90 mg / mL or less, even more preferably 85 mg / mL or less, still even more preferably 80 mg / mL or less, particularly preferably 75 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, or 50 mg / mL or less. More specifically, the concentration of sugar varies depending on factors such as the type of sugar. For example, it may be 2.7 - 800 mg / mL, 2.7 - 600 mg / mL, 2.7 - 400 mg / mL, 2.7 - 200 mg / mL, or 2.7 - 100 mg / mL, preferably 3.0 - 95 mg / mL, more preferably 3.5 - 90 mg / mL, even more preferably 4.0 - 85 mg / mL, still even more preferably 4.5 - 80 mg / mL, particularly preferably 5.0 - 75 mg / mL, 6.0 - 70 mg / mL, 8.0 - 60 mg / mL, or 10.0 - 50 mg / mL.

[0053] The concentration of the chelating agent in the composition is not particularly limited as long as it can stabilize the extracellular vesicles after being mixed with the extracellular vesicle-containing sample. Such a concentration varies depending on factors such as the type of chelating agent, but for example, it may be 1.1 mM or more, preferably 5 mM or more, preferably 10 mM or more, preferably 15 mM or more, more preferably 20 mM or more, even more preferably 30 mM or more, still even more preferably 40 mM or more, and particularly preferably 50 mM or more. Such a concentration also varies depending on factors such as the type of chelating agent, but for example, it may be 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, even more preferably 160 mM or less, still even more preferably 150 mM or less, particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less. More specifically, the concentration of the chelating agent may be, for example, 1.1 mM to 200 mM, preferably 5 to 180 mM, more preferably 10 to 170 mM, even more preferably 15 to 160 mM, still even more preferably 20 to 150 mM, particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

[0054] When the composition of the present invention contains both a sugar and a chelating agent, the concentrations of the sugar and the chelating agent can also be defined by their ratio. For example, the concentration of the chelating agent per 10 mg / mL of the sugar varies depending on factors such as the types of the sugar and the chelating agent, but is, for example, 1 mM or more, preferably 5 mM or more, preferably 10 mM or more, preferably 15 mM or more, more preferably 20 mM or more, even more preferably 30 mM or more, still even more preferably 40 mM or more, and particularly preferably 50 mM or more. Such a concentration also varies depending on factors such as the types of the sugar and the chelating agent, but is, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, even more preferably 160 mM or less, still even more preferably 150 mM or less, particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less. More specifically, the concentration of the chelating agent may be, for example, 1 mM to 200 mM, preferably 5 to 180 mM, more preferably 10 to 170 mM, even more preferably 15 to 160 mM, still even more preferably 20 to 150 mM, particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.

[0055] The composition of the present invention may be a non-aqueous composition (e.g., a mixed powder) containing a sugar and / or a chelating agent, which is dissolved in an aqueous solution during use, or an aqueous solution containing a sugar and / or a chelating agent. However, from the viewpoints of quick and convenient use, etc., an aqueous solution containing a sugar and / or a chelating agent is preferred. Examples of the aqueous solution include water (e.g., distilled water, sterilized water, sterilized distilled water, and pure water), as well as buffer solutions, with buffer solutions being preferred. Examples of buffer solutions include phosphate buffer, phosphate buffered saline (PBS), tartrate buffer, citrate buffer, acetate buffer, glycine buffer, carbonate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, boric acid buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, N,N-bis(2-hydroxyethyl)glycine (Bis-Tris) buffer, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer. The pH of the buffer solution is preferably neutral. More specifically, such a pH may preferably be 5.0 or higher, more preferably 5.5 or higher, and even more preferably 6.0 or higher. The pH may also preferably be 9.0 or lower, more preferably 8.5 or lower, and even more preferably 8.0 or lower. The pH can be measured using a known method in the art. Preferably, the pH can adopt the value measured at 25 °C using a pH meter having a glass electrode.

[0056] The composition of the present invention can be appropriately mixed with a sample containing extracellular vesicles and used. The mixing ratio of the composition of the present invention to the sample containing extracellular vesicles (sample containing extracellular vesicles / composition) is, for example, 1 / 30 to 30, preferably 1 / 20 to 20, more preferably 1 / 10 to 10, and even more preferably 1 / 10 to 1.

[0057] When the composition of the present invention is an aqueous solution, the volume of the aqueous solution is, for example, 1 μL to 100 mL. Preferably, the volume of the aqueous solution may be 10 μL or more, 100 μL or more, or 1000 μL or more. The volume of the aqueous solution may also be 50 mL or less, 10 mL or less, or 2 mL or less.

[0058] In another embodiment, the reagent of the present invention is a kit containing a sugar and / or a chelating agent. The sugar and / or the chelating agent can be provided in solid or aqueous solution form, but is preferably provided in aqueous solution form. Accordingly, the kit of the present invention may be provided in the form of an aqueous solution containing a sugar, in the form of an aqueous solution containing a chelating agent, in the form of an aqueous solution containing a sugar and a chelating agent, or in the form of a first aqueous solution containing a sugar and a second aqueous solution containing a chelating agent. The aqueous solution is as described above, but a buffer solution is preferred. Examples of buffer solutions and pH are as described above. The relationship between the concentration of the sugar in the first aqueous solution, the concentration of the chelating agent in the second aqueous solution, and the concentration ratio of the sugar and the chelating agent in the first and second aqueous solutions is the same as that described above for the composition of the present invention. Also, the mixing ratio of the first and second aqueous solutions with the extracellular vesicle-containing sample and the volume of each of the first and second aqueous solutions are the same as those described above for the composition of the present invention.

[0059] 3. Mixture containing extracellular vesicles The present invention also provides a mixture containing a sugar and / or a chelating agent and extracellular vesicles. The mixture of the present invention may further contain an aqueous solution (e.g., a buffer solution as described above). The form of the mixture is not particularly limited, but an aqueous solution or its frozen product (e.g., freeze-dried product) is preferred. The mixture of the present invention can be obtained by treating an extracellular vesicle-containing sample with the method or reagent of the present invention as described above. The mixture of the present invention is useful, for example, for the preservation of extracellular vesicles.

[0060] The concentration of sugar or chelating agent in the mixture, as well as the concentration ratio of sugar and chelating agent, are the same as those described above for the method of the present invention. The concentration of extracellular vesicles (number of particles / mL) in the mixture is, for example, 1×10 2 ~1×10 15 Preferably 1×10 3 ~1×10 14 More preferably 1×10 4 ~1×10 13 Even more preferably 1×10 5 ~1×10 12 Particularly preferably 1×10 6 ~1×10 11 .

[0061] When the mixture of the present invention is an aqueous solution, the volume of the aqueous solution is, for example, 1 μL to 100 mL. Preferably, the volume of the aqueous solution may be 10 μL or more, 100 μL or more, or 1000 μL or more. The volume of the aqueous solution may also be 50 mL or less, 10 mL or less, or 2 mL or less.

Examples

[0062] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0063] Example 1: Stabilization of exosomes by chelating agent The effect of chelating agent on the lyophilization of exosomes was examined.

[0064] 1) Recovery of exosomes The culture supernatant of human non-small cell lung cancer cell H1299 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000×g for 5 minutes at 4°C, filtered through a 0.22 μm filter (manufactured by Millipore), and then concentrated using Amicon Ultra-15 (manufactured by Millipore). The concentrate was centrifuged at 20,000×g for 15 minutes at 4°C. Next, the supernatant was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, and PBS (2.9 mM NaH2PO4, 9.0 mM Na2HPO4, 137 mM NaCl) or 50 mM EDTA / 50 mM EGTA / PBS was added to resuspend the precipitate. After resuspension, it was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, and fresh PBS or 50 mM EDTA / 50 mM EGTA / PBS (recovery buffer) was added to resuspend the precipitate and recover exosomes.

[0065] 2) Lyophilization of exosomes The recovered exosomes were quantified for protein using the Qubit™ Protein Assay Kit (manufactured by Thermo Fisher Scientific), and the recovery buffer was further added to each of the recovered exosomes to prepare them to contain the same concentration of protein. Next, the recovered exosomes were mixed with the lyophilization buffer so that the volume ratio of the recovered exosomes to the lyophilization buffer was 1:24, dispensed, and then lyophilized using a lyophilizer (manufactured by Tokyo Rika Kikai Co., Ltd.) at -28°C for 2 hours, -10°C for 4 hours, and then at 20°C to obtain a lyophilized product containing lyophilized exosomes. As the lyophilization buffer, PBS or EDTA / EGTA / PBS (final conc. 50 mM EDTA / 50 mM EGTA) was used. The pH of the PBS used was 7.4 (the same in Examples 1 to 3). Thereafter, the lyophilized product was dissolved in Milli-Q water (manufactured by Millipore) or 50 mM EDTA / 50 mM EGTA / H2O (dissolution buffer).

[0066] 3) Evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) The amount of these lyophilized exosomes in solution was evaluated by ELISA. Specifically, PBS (pH 7.4) containing the in-house anti-CD9 antibody was added to a 96-well ELISA plate (manufactured by NUNC) and incubated overnight at 4°C. Thereafter, each well was washed three times with PBS containing 0.05 wt% Tween (registered trademark) 20 (PBS-T), 200 μL of PBS containing 0.5 wt% casein was added, and the mixture was incubated at room temperature for 2 hours. After washing with PBS-T, 100 μL of the lysate of the lyophilized product diluted with PBS was added to each well and incubated at 37°C for 1 hour. Thereafter, after washing with PBS-T, 100 μL of PBS containing the biotinylated in-house anti-CD9 antibody and streptavidin-conjugated alkaline phosphatase (SA-ALP, manufactured by GeneTex) was added to each well and incubated at 37°C for 1 hour. Thereafter, the wells were washed with PBS, 100 μL of the Lumipulse (registered trademark) substrate solution (manufactured by Fuji Rebio) was added, the reaction was carried out at 37°C for 10 minutes, and the luminescence count at a wavelength of 477 nm was measured. The count of the non-lyophilized sample was taken as 100%. As a result, in the lyophilization buffer containing the chelating agent, the amount of exosomes increased (Figure 1). Therefore, it was shown that the chelating agent can stabilize exosomes during lyophilization of exosomes.

[0067] Example 2: Stabilization of exosomes by sucrose The effect of sugar on the lyophilization of exosomes was investigated.

[0068] 1) Recovery of exosomes The culture supernatant of human colon cancer cells SW480 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000×g for 5 minutes at 4°C, filtered through a 0.22-μm filter (Millipore), and then concentrated using Amicon Ultra-15 (Millipore). The concentrate was centrifuged at 20,000×g for 15 minutes at 4°C. Next, the supernatant was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, PBS was added, and the precipitate was resuspended. After resuspension, it was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, fresh PBS was added, and the precipitate was resuspended to recover exosomes.

[0069] 2) Lyophilization of exosomes The recovered exosomes were quantified for protein using the Qubit (trademark) Protein Assay Kit (Thermo Fisher Scientific), and PBS was further added to each of the recovered exosomes to prepare them to contain the same concentration of protein. Next, the recovered exosomes were mixed with the lyophilization buffer so that the volume ratio of the recovered exosomes to the lyophilization buffer was 1:24, dispensed, and then lyophilized at -28°C for 2 hours, at -10°C for 4 hours, and then using a 20°C program to obtain a lyophilized product containing lyophilized exosomes. Note that as the lyophilization buffer, PBS, EDTA / EGTA / PBS (final conc. 50 mM EDTA / 50 mM EGTA), sucrose / PBS (final conc. 10 mg / mL sucrose), or EDTA / EGTA / sucrose / PBS (final conc. 50 mM EDTA / 50 mM EGTA / 10 mg / mL sucrose) was used. Thereafter, the lyophilized product was dissolved in Milli-Q water (Millipore).

[0070] 3) Evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) The amount of these lyophilized exosomes in solution was evaluated by ELISA. Specifically, PBS (pH 7.4) containing the in-house anti-CD9 antibody was added to a 96-well ELISA plate (manufactured by NUNC) and incubated overnight at 4°C. Thereafter, each well was washed three times with PBS-T, 200 μL of PBS containing 0.5 wt% Casein was added, and the mixture was incubated at room temperature for 2 hours. After washing with PBS-T, 100 μL of the lysate of the lyophilized product diluted with PBS was added to each well and incubated at 37°C for 1 hour. Thereafter, after washing with PBS-T, 100 μL of PBS containing the biotinylated in-house anti-CD9 antibody and streptavidin-conjugated alkaline phosphatase (SA-ALP, manufactured by GeneTex) was added to each well and incubated at 37°C for 1 hour. Thereafter, the wells were washed with PBS, 100 μL of the lumipulse substrate solution was added, and after reacting at 37°C for 5 minutes, the luminescence count at a wavelength of 477 nm was measured. The count of the non-lyophilized sample was set as 100%. As a result, in the lyophilization buffer containing sucrose, the amount of exosomes increased (Figure 2). Also, in the lyophilization buffer containing sucrose and a chelating agent, the amount of exosomes further increased. Therefore, it was shown that sugar can stabilize exosomes in the lyophilization of exosomes. Also, it was shown that the stabilization of exosomes by sugar is improved in the presence of a chelating agent.

[0071] Example 3: Evaluation of the stability of lyophilized exosomes based on particle amount measurement The lyophilized samples were measured with NanoSight, and the stability was evaluated based on the particle amount of the lyophilized exosomes.

[0072] 1) Recovery of exosomes The culture supernatant of human colon cancer cells SW1116 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000×g for 5 minutes at 4°C, filtered through a 0.22-μm filter (Millipore), and then concentrated using Amicon Ultra-15 (Millipore). The concentrate was centrifuged at 20,000×g for 15 minutes at 4°C. Next, the supernatant was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, and PBS (Control) or 50 mM EDTA / 50 mM EGTA / PBS (ED / EG) was added to resuspend the precipitate. After resuspension, it was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, and fresh PBS or 50 mM EDTA / 50 mM EGTA / PBS (ED / EG) was added to resuspend the precipitate to recover exosomes.

[0073] 2) Lyophilization of exosomes The recovered exosomes were quantified for protein using the Qubit™ Protein Assay Kit (Thermo Fisher Scientific), and recovery buffer was further added to each of the recovered exosomes to prepare them to contain the same concentration of protein. Next, the recovered exosomes were mixed with the lyophilization buffer so that the volume ratio of the recovered exosomes to the lyophilization buffer was 1:24, dispensed, and then lyophilized at -28°C for 2 hours, at -10°C for 4 hours, and then using a 20°C program using the above-described lyophilizer to obtain a lyophilized product containing lyophilized exosomes. PBS and EDTA / EGTA / PBS (final conc. 50 mM EDTA / 50 mM EGTA) (ED / EG) were used as the lyophilization buffer. Thereafter, the lyophilized product was dissolved in Milli-Q water (Millipore).

[0074] 3) Stability evaluation of lyophilized exosomes The number and distribution of the dissolved lyophilized exosomes were measured using NanoSight LM10 (Malvern Instruments). The measurement was performed with a measurement time of 30 seconds and a repetition number of 3 times, and analyzed with a detection threshold of 15. As a result, in the lyophilized buffer containing the chelating agent, the amount of particles in the range of 100 to 200 nm, which is considered to correspond to exosomes, increased (Table 1 and Figures 3A to 3D). Therefore, it was also shown by the measurement of the amount of particles that the chelating agent can stabilize exosomes during lyophilization.

[0075] [Table 1]

[0076] Reference Example 1: Treatment of exosomes with various chelating agents As chelating agents, disodium ethylenediaminetetraacetate (EDTA·2Na), ethylene glycol ether diamine tetraacetic acid (EGTA), hydroxyethylethylenediamine triacetic acid (HEDTA), hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid dihydrate, ethylenediamine tetra(methylenephosphonic acid) (EDTMP) (EDTA·2Na and EGTA were manufactured by Dojindo Laboratories, HEDTA, HIDA, NTA, and EDTMP were manufactured by Tokyo Chemical Industry Co., Ltd., and oxalic acid dihydrate was manufactured by Wako Pure Chemical Industries, Ltd.) were used. The effects of each chelating agent on the sedimentation amount of exosomes were examined. 300 μL of serum specimen or plasma specimen was mixed with 600 μL of PBS or PBS supplemented with each chelating agent. The concentrations of each chelating agent were used to be 1, 10, and 50 mM at the final concentration (however, EDTMP was only 1, 10, and 30 mM). Then, after leaving the solution at room temperature for 30 minutes, it was centrifuged at 20,000×g at 4°C for 15 minutes. The supernatant was transferred to a new tube, and magnetic beads (Protein G Dynabeads) fixed with 2 μg of a monoclonal antibody recognizing CD9 were added thereto. After reacting overnight at 4°C, the magnetic beads were washed 3 times with PBS, and the sample was eluted from the magnetic beads with sample buffer (containing SDS) to obtain a sample for Western blotting. These samples were analyzed by Western blotting with a biotinylated anti-CD9 antibody. As a result, in both serum (Figure 4A) and plasma (Figure 4B), all of the chelating agents used showed the recovery of exosomes by immunoprecipitation, and the amount of immunoprecipitated exosomes increased as the concentration increased. In particular, with 30 mM EDTMP, 50 mM EDTA, EGTA, HEDTA, NTA, and oxalic acid, the amount of immunoprecipitated exosomes increased significantly (Figure 4A, 4B).

[0077] Example 4: Stabilization of Exosomes by Various Sugars The effects of various sugars on the lyophilization of exosomes were examined.

[0078] 1) Recovery of Exosomes The culture supernatant of human non-small cell lung cancer cell H1299 cultured in serum-free medium for 3 days was used as a sample, and exosomes were recovered by the recovery method of Example 2.

[0079] 2) Lyophilization of Exosomes The recovered exosomes were quantified for protein using the Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific), and PBS was further added to each of the recovered exosomes to prepare them to contain the same concentration of protein. Next, the recovered exosomes were mixed with the lyophilization buffer so that the volume ratio of the recovered exosomes to the lyophilization buffer was 1:9. After dispensing, the mixture was lyophilized using a lyophilizer (manufactured by Genevac) to obtain a lyophilized product containing lyophilized exosomes. As the lyophilization buffer, PBS, and lactose, fructose, methyl-β-cyclodextrin (MβCD), hydroxypropyl-β-cyclodextrin (HpβCD), γ-cyclodextrin (γCD), glucose, lactitol, sorbitol, xylitol, sucrose, trehalose, mannitol, and xylose were used at final concentrations of 2.0 mg / mL, 10 mg / mL, and 50 mg / mL. Thereafter, the lyophilized product was dissolved in Milli-Q water (manufactured by Millipore).

[0080] 3) Evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) The amount of these lyophilized exosomes in solution was evaluated using an ELISA system. Specifically, PBS (pH 7.4) containing the in-house anti-CD9 antibody was added to a 96-well ELISA plate (manufactured by NUNC) and incubated overnight at 4°C. Subsequently, each well was washed three times with PBS-T, 200 μL of PBS containing 0.5 wt% BSA was added, and the mixture was incubated at room temperature for 2 hours. After washing with PBS-T, 100 μL of the lysate of the lyophilized product was added to each well and incubated at 37°C for 1 hour. Then, after washing with PBS-T, 100 μL of PBS containing the biotinylated in-house anti-CD9 antibody was added to each well and incubated at 37°C for 1 hour. Then, after washing with PBS-T, 100 μL of PBS containing streptavidin-conjugated alkaline phosphatase (SA-ALP, manufactured by GeneTex) was added to each well and incubated at 37°C for 1 hour. Then, after washing with PBS-T, 100 μL of the Lumi-Pulse substrate solution was added, and after reacting at 37°C for 5 minutes, the luminescence count at a wavelength of 480 nm was measured. The count of the non-lyophilized sample was set as 100%. As a result, in these lyophilization buffers containing saccharides, the amount of exosomes increased (FIGS. 5A and 5B). In particular, in the lyophilization buffers containing sugar at 10 mg / mL or 50 mg / mL, the amount of exosomes increased with most of the sugars examined. Therefore, it was shown that various sugars can stabilize exosomes in the lyophilization of exosomes, and particularly a high stabilization effect is achieved when the sugar concentration is 10 to 50 mg / mL.

[0081] Example 5: Stabilization of exosomes by combined use of various sugars and chelating agents The effect of the combined use of various sugars and chelating agents on the lyophilization of exosomes was examined.

[0082] 1) Recovery of exosomes, lyophilization of exosomes, and evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) Except for the lyophilization buffer, exosome recovery, lyophilization, and exosome stability evaluation were performed by the method described in Example 4. As the lyophilization buffer, PBS or EDTA / EGTA / PBS (final conc. 50 mM EDTA / 50 mM EGTA) (ED / EG), or a solution in which lactose, γ-cyclodextrin (γCD), glucose, or lactitol was added to either of these at a final concentration of 10 mg / mL was used.

[0083] As a result, the amount of exosomes increased in the lyophilization buffers containing these saccharides and chelating agents (Figure 6). Therefore, it was shown that the stabilization of exosomes by sugars was improved in the presence of chelating agents.

[0084] Example 6: Stabilization of serum-derived exosomes The effects of various saccharides, chelating agents, or their combinations on the lyophilization of serum-derived exosomes were examined.

[0085] 1) Exosome recovery Human serum was used as a sample. The serum was centrifuged at 2,000×g for 5 minutes at 4°C, and then at 20,000×g for 15 minutes at 4°C. Next, the supernatant was centrifuged at 100,000×g for 3 hours at 4°C. The supernatant was discarded, PBS was added, and the precipitate was resuspended. After resuspension, it was centrifuged at 100,000×g for 1 hour at 4°C. The supernatant was discarded, fresh PBS was added, and the precipitate was resuspended to recover exosomes.

[0086] 2) Lyophilization of exosomes and evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) Lyophilization of exosomes and evaluation of exosome stability were performed by the method described in Example 4, except for the lyophilization buffer. As the lyophilization buffer, PBS or EDTA / EGTA / PBS (final conc. 50 mM EDTA / 50 mM EGTA) (ED / EG), or those obtained by adding lactose, glucose, or lactitol to any of these at a final conc. of 10 mg / mL or 50 mg / mL were used.

[0087] As a result, in the lyophilization buffer containing these saccharides or chelating agents, the amount of exosomes increased (Figure 7). Furthermore, when using a lyophilization buffer containing both saccharides and a chelating agent, the amount of exosomes further increased. Therefore, the effect of improving the stabilization of exosomes by saccharides and chelating agents was also shown in serum-derived exosomes.

[0088] Example 7: Stabilization of exosomes by carboxymethylcellulose The effect of carboxymethylcellulose (CMC) on the lyophilization of exosomes was examined.

[0089] 1) Recovery of exosomes The culture supernatant of human non-small cell lung cancer cell H1299 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000×g at 4°C for 5 minutes, filtered through a 0.22-μm filter (manufactured by Millipore), and then concentrated using Amicon Ultra-15 (manufactured by Millipore). The concentrate was centrifuged at 100,000×g at 4°C for 3 hours. The supernatant was discarded, PBS was added, and the precipitate was resuspended. After resuspension, it was centrifuged at 100,000×g at 4°C for 1 hour. The supernatant was discarded, and fresh PBS was added to recover exosomes.

[0090] 2) Lyophilization of exosomes The recovered exosomes were quantified for protein using the Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific), and the concentration was adjusted. Next, an equal volume of lyophilization buffer was mixed with 1 volume of the recovered exosomes, frozen at -20°C for 3 hours, and then dried using a centrifugal evaporator (manufactured by Scrum). Thereafter, the lyophilized product was dissolved in Milli-Q water (manufactured by Millipore), and further diluted by adding PBS. PBS, CMC / PBS (final conc. 0.2 wt% or 1 wt% CMC) was used as the lyophilization buffer.

[0091] 3) Evaluation of exosome stability based on measurement of the amount of exosome-specific antigen (CD9) The amount of these dissolved lyophilized exosomes was evaluated with an ELISA system using the in-house anti-CD9 antibody immobilized on a solid phase, the in-house biotinylated anti-CD9 antibody for detection, and SA-ALP (manufactured by GeneTex), with the count of the non-lyophilized sample set as 100%. As a result, the amount of remaining exosomes increased under the conditions containing 0.2 wt% CMC or 1 wt% CMC during lyophilization (Figure 8). Therefore, it was shown that CMC can stabilize exosomes during lyophilization.

Claims

1. The following: (1) Mixing a sample containing extracellular vesicles with a sugar and a chelating agent; and (2) Lyophilizing the mixture of the sample containing extracellular vesicles, the sugar, and the chelating agent, wherein the concentration of the chelating agent in the mixing is 40 to 200 mM, the chelating agent is one or more selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), glycol ether diamine tetraacetic acid (EGTA), hydroxyethylethylenediamine triacetic acid (HEDTA), hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediamine tetra(methylenephosphonic acid) (EDTMP), and salts thereof, A method for stabilizing the freeze-drying preservation of extracellular vesicles.

2. The method according to claim 1, wherein the extracellular vesicles are exosomes.

3. The method according to claim 1 or 2, wherein the sample containing extracellular vesicles is a body fluid or a culture supernatant.

4. The method according to any one of claims 1 to 3, wherein the sample containing extracellular vesicles is a blood sample.

5. The method according to any one of claims 1 to 4, wherein the concentration of the sugar in the mixing is 2.5 to 100 mg / mL.

6. The method according to any one of claims 1 to 5, wherein the concentration of the chelating agent in the mixing is 40 to 120 mM.

7. The method according to any one of claims 1 to 6, wherein the concentration of the chelating agent in the mixing is 50 to 100 mM.

8. The method according to any one of claims 1 to 7, wherein the concentration of the chelating agent per 10 mg / mL of the sugar is 1 mM or more.

9. The method according to any one of claims 1 to 8, comprising the following: (1') When the sample containing extracellular vesicles is a biological liquid sample containing extracellular vesicles, recovering the extracellular vesicles from the biological liquid sample containing extracellular vesicles; (2') Mixing the recovered extracellular vesicles with a sugar and a chelating agent in a solution to prepare a solution containing extracellular vesicles, the sugar, and the chelating agent, wherein the concentration of the chelating agent is 40 to 200 mM; and (3') Lyophilizing the solution containing extracellular vesicles, the sugar, and the chelating agent to prepare a lyophilized product of extracellular vesicles, the sugar, and the chelating agent.

10. The method according to claim 9, wherein the recovery of the extracellular vesicles is performed as follows: (a) Mixing a biological liquid sample containing extracellular vesicles with a chelating agent to prepare a biological liquid sample containing extracellular vesicles and the chelating agent; (b) Recovering extracellular vesicles as a precipitate from the biological liquid sample containing extracellular vesicles and the chelating agent. **Claim 11** Comprising a sugar and a chelating agent, wherein the sugar and the chelating agent are used for being mixed with extracellular vesicles in a solution, the concentration of the chelating agent in the mixing is 40 to 200 mM, the chelating agent is one or more selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), glycol ether diamine tetraacetic acid (EGTA), hydroxyethylethylenediamine triacetic acid (HEDTA), hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediamine tetra(methylenephosphonic acid) (EDTMP), and salts thereof, A reagent for freeze-drying preservation stabilization of extracellular vesicles. **Claim 12** The reagent according to claim 11, wherein the concentration of the sugar in the mixing is 2.5 to 100 mg / mL. **Claim 13** The reagent according to claim 11 or 12, wherein the concentration of the chelating agent per 10 mg / mL of the sugar is 1 mM or more.

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