US20200319175A1 - Method for stabilizing extracellular vesicles - Google Patents

Method for stabilizing extracellular vesicles Download PDF

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US20200319175A1
US20200319175A1 US16/955,553 US201816955553A US2020319175A1 US 20200319175 A1 US20200319175 A1 US 20200319175A1 US 201816955553 A US201816955553 A US 201816955553A US 2020319175 A1 US2020319175 A1 US 2020319175A1
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extracellular vesicle
freeze
saccharide
chelating agent
exosomes
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Tatsutoshi INUZUKA
Ayako Kurimoto
Yuki KAWASAKI
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HU Group Research Institute GK
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Miraca Research Institute GK
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Assigned to H.U. GROUP RESEARCH INSTITUTE G.K. reassignment H.U. GROUP RESEARCH INSTITUTE G.K. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: MIRACA RESEARCH INSTITUTE G.K.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

  • the present invention relates to a method of stabilizing extracellular vesicle(s), and the like.
  • An extracellular vesicle is a microscopic vesicle secreted from various types of cells and having a membrane structure, and exists in body fluids such as blood.
  • the extracellular vesicles secreted extracellularly include exosomes, ectosomes, and apoptotic blebs. Since the extracellular vesicle contains various substances that play a function such as intercellular signaling, it has been analyzed for the purposes of diagnosis, drug discovery and the like. Thus, it is required to develop a method of treating the extracellular vesicles useful for such analyses.
  • Patent Literature 1 describes: (1) capability of improving a yield of extracellular vesicles in the presence of EDTA (chelating agent) at a low concentration of approximately 3.0 mg/mL (approximately 10 ⁇ M) or less and incapability of improving the yield of extracellular vesicles in the presence of EDTA at a higher concentration (Example 1 and FIGS. 1A to 1C ); (2) preserving extracellular vesicles in the presence of EDTA at low concentration of approximately 2.25 mg/ml (approximately 7.7 ⁇ M) at a certain temperature and time (Example 2, FIGS.
  • EDTA chelating agent
  • Patent Literature 1 US Patent Application Publication No. 2015/0125864
  • extracellular vesicles can be stabilized, such extracellular vesicles are promising for improvement in preservability thereof and application to diagnosis, drug discovery and the like. Therefore, it is an object of the present invention to develop a method for stabilizing extracellular vesicle(s).
  • extracellular vesicle(s) in the extracellular vesicle-containing sample can be stabilized by mixing the extracellular vesicle-containing sample with a certain component such as a saccharide, and completed the present invention.
  • the present invention is as follows.
  • a method of stabilizing an extracellular vesicle comprising mixing an extracellular vesicle-containing sample with a saccharide and a chelating agent.
  • the extracellular vesicle-containing sample is a body fluid or a culture supernatant.
  • the method according to any of [1] to [3], wherein the extracellular vesicle-containing sample is a blood sample.
  • a concentration of the saccharide in the mixing is 2.5 to 100 mg/mL.
  • a method of stabilizing an extracellular vesicle comprising: (1) mixing an extracellular vesicle-containing sample with a saccharide; and (2) freezing a mixture of the extracellular vesicle-containing sample and the saccharide.
  • the freezing is freeze-drying.
  • a method of stabilizing an extracellular vesicle comprising: (1) mixing an extracellular vesicle-containing sample with a chelating agent; and (2) freezing a mixture of the extracellular vesicle-containing sample and the chelating agent.
  • the extracellular vesicle(s) can be stabilized in the extracellular vesicle-containing sample by mixing the extracellular vesicle-containing sample with the saccharide. Therefore, the present invention is useful, for example, in preservation of the extracellular vesicle(s).
  • FIG. 1 includes graphs of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 1. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • FIG. 2 is a graph of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 2. Yields of exosomes obtained without freeze-drying are defined as 100.
  • FIG. 3A is a graph of particle counts and a particle diameter distribution measured in a sample (Control) that is obtained before freeze-drying a culture supernatant of a human colon carcinoma cell line SW1116 in Example 3.
  • FIG. 3B is a graph of particle counts and a particle diameter distribution measured in a sample (ED/EG) that is obtained before freeze-drying the culture supernatant of the human colon carcinoma cell line SW1116 in Example 3.
  • FIG. 3C is a graph of particle counts and a particle diameter distribution measured in a sample (Control) that is obtained after freeze-drying the culture supernatant of the human colon carcinoma cell line SW1116 in Example 3.
  • FIG. 3D is a graph of particle counts and a particle diameter distribution measured in a sample (ED/EG) that is obtained after freeze-drying the culture supernatant of the human colon carcinoma cell line SW1116 in Example 3.
  • FIG. 4A is a diagram depicting results of western blotting with a biotinylated anti-CD9 antibody of samples obtained from an immunoprecipitation method of a serum specimen that is mixed with each of various chelating agents at different concentrations in Reference Example 1.
  • FIG. 4B is a diagram depicting results of western blotting with the biotinylated anti-CD9 antibody of samples obtained from the immunoprecipitation method of a plasma specimen that is mixed with each of various chelating agents at different concentrations in Reference Example 1.
  • FIG. 5A is a graph (continued on FIG. 5B ) of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 4. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • FIG. 5B is a graph of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 4. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • FIG. 6 is a graph of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 5. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • FIG. 7 is a graph of yields of exosomes obtained after freeze-drying a human serum sample under various buffer conditions in Example 6. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • FIG. 8 is a graph of yields of exosomes obtained after freeze-drying under various buffer conditions in Example 7. Yields of exosomes obtained without freeze-drying are defined as 100%.
  • the present invention provides a method of stabilizing an extracellular vesicle, including mixing an extracellular vesicle-containing sample with a saccharide.
  • the invention also provides the method of stabilizing the extracellular vesicle, including mixing the extracellular vesicle-containing sample with a chelating agent.
  • the invention also provides the method of stabilizing the extracellular vesicle, including mixing the extracellular vesicle-containing sample with the saccharide and the chelating agent.
  • the extracellular vesicle is a microscopic vesicle secreted from various types of cells and having a membrane structure.
  • Examples of the extracellular vesicle include exosomes, ectosomes and apoptotic blebs.
  • the extracellular vesicle is the exosome.
  • the extracellular vesicle can also be defined by its size. The size of the extracellular vesicle is, for example, 30 to 1000 nm, preferably 50 to 300 nm, and more preferably 80 to 200 nm.
  • the size of the extracellular vesicle can be measured by, for example, a method based on Brownian movement of the extracellular vesicle, a light scattering method, and an electric resistance method, and the like.
  • the size of the extracellular vesicle is measured by NanoSight LM10 (manufactured by Malvern Instruments). In the case of using the NanoSight LM10, a measurement time of 30 seconds, three repetition times, and a detection threshold of 15 can be employed as measurement conditions.
  • the extracellular vesicle can also be defined by using an extracellular vesicle marker.
  • extracellular vesicle marker examples 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 and EpCAM.
  • CCA carcinoembryonic antigen
  • HSP heat shock protein
  • MHC major histocompatibility complex
  • TSG tumor susceptibility gene
  • LAMP lysosome associated membrane protein
  • IAM intercellular adhesion molecule
  • integrin integrin
  • ceramide cholesterol
  • cholesterol phosphatidylserine
  • ALIX Annexins
  • Caveolin-I Caveolin-I
  • the extracellular vesicle-containing sample is any sample that contains the extracellular vesicle.
  • the extracellular vesicle-containing sample is a biological liquid sample.
  • the extracellular vesicle-containing sample may be subjected to another treatment before being used for the method of the present invention. Examples of such a treatment include centrifugation, extraction, filtration, precipitation, heating, refrigeration, and stirring.
  • the extracellular vesicle-containing sample is a culture supernatant.
  • the culture supernatant may be a cell culture supernatant or a tissue culture supernatant.
  • the organism from which a cell or a tissue to be cultured is derived include animals such as mammalian animals (e.g., primates such as humans and monkeys; rodents such as mice, rats and rabbits; farm animals such as cattle, pigs and goats; and working animals such as horses and sheep) and birds (e.g., chickens), insects, microorganisms (e.g., bacteria), plants and fish.
  • the organisms are mammalian animals such as humans.
  • the extracellular vesicle-containing sample is a body fluid.
  • the body fluid is derived from the organism as described above.
  • the body fluid include blood samples (e.g., whole blood, serum and plasma), lymph fluid, tissue fluid, cerebrospinal fluid, ascites, saliva, pancreatic liquid, bile, sweat, seminal fluid, urine, tear fluid, mucosal fluid, breast fluid, thoracic fluid, bronchoalveolar lavage fluid and amnion fluid.
  • the body fluid is the blood.
  • the extracellular vesicle-containing sample is a milk or a fruit juice.
  • the saccharide is a monosaccharide (e.g., aldose and ketose) or a polysaccharide in which two or more monosaccharides are linked by a glycosidic bond.
  • the saccharides also include derivatives and saccharide alcohols.
  • the derivative of the saccharide refers to a compound in which a hydrogen atom, a hydroxy group or a carbonyl group is substituted with a substituent in the saccharide.
  • the saccharide alcohol refers to a compound in which the carbonyl group is reduced in the saccharide.
  • examples of the monosaccharide include trioses (e.g., glyceraldehyde, dihydroxyacetone), tetroses (e.g., erythrose, threose, erythrose), 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, coriose).
  • trioses e.g., glyceraldehyde, dihydroxyacetone
  • tetroses e.g., erythrose, threose, erythrose
  • pentoses
  • aldose examples include xylose, glucose, galactose, mannose, glyceraldehyde, erythrose, threose, ribose, arabinose, lyxose, allose, altrose, gulose, idose, and talose.
  • ketose examples include fructose, dihydroxyacetone, erythrulose, xylulose, ribulose, psicose, sorbose, tagatose, sedoheptulose and coriose.
  • polysaccharide examples include the polysaccharide in which two or more of the monosaccharides as described above are linked together.
  • examples of the polysaccharide include polysaccharides in which two or more monosaccharide units in one or plural kinds of are linked by a glycosidic bond (e.g., one or more glucose and one or more fructose are linked by a glycosidic bond), and the monosaccharide units are selected from the group consisting of glucose, fructose, galactose, mannose and xylose.
  • a polysaccharide may be a linear polysaccharide or a cyclic polysaccharide.
  • the linear polysaccharide may be a linear oligosaccharide or a linear polymer polysaccharide.
  • 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 still more preferably 2 to 4.
  • each of the numbers of glucose and fructose in the polysaccharide may be, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and still more preferably 1 or 2.
  • the linear oligosaccharide may be a disaccharide, for example.
  • the disaccharide include sucrose, lactose, and trehalose.
  • the disaccharide is sucrose in which one glucose and one fructose are linked by a glycosidic bond.
  • linear polymer polysaccharide examples include cellulose, amylose, amylopectin, glucomannan, pullulan, galactomannan, inulin, glycogen, chitin, chitosan, glucuronoxylan, arabinoxylan, agarose, carrageenan, pectin, pectinic acid, alginic acid, fucoidan, chondroitin sulfate and hyaluronan.
  • the polysaccharide is the 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 still more preferably 6 to 8.
  • the cyclic polysaccharide examples include cyclodextrins ( ⁇ -cyclodextrin, ⁇ -cyclodextrin and ⁇ -cyclodextrin).
  • Examples of the substituent in the saccharide derivative include a hydrogen atom, a hydroxy group, a C 1-6 alkyl group, a C 1-6 alkenyl group, a C 1-6 alkynyl group, a C 1-6 alkyloxy (alkoxy) group, a C 6-14 aromatic hydrocarbon group, a C 1-6 alkyl-carbonyl (acyl) group, a carboxy group, a nitro group, an amino group and a cyano group.
  • the C 1-6 alkyl group is an alkyl having one to six carbon atom(s), and may be linear, branched or cyclic, and the linear or branched alkyl is preferable.
  • Examples of the C 1-6 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.
  • the C 1-6 alkyl group is preferably a C 1-4 alkyl group, and more preferably a C 1-3 alkyl group.
  • the C 1-6 alkenyl group is an alkenyl group having one to six carbon atom(s), and may be linear, branched or cyclic, and the linear or branched alkenyl is preferable.
  • Examples of the C 1-6 alkenyl group include ethenyl (vinyl), propenyl, butenyl, pentenyl and hexenyl.
  • the C 1-6 alkenyl group is preferably a C 1-4 alkenyl group, and more preferably a C 1-3 alkenyl group.
  • the C 1-6 alkynyl group is an alkynyl group having one to six carbon atom(s), and may be linear, branched or cyclic, and the linear or branched alkynyl is preferable.
  • Examples of the C 1-6 alkynyl group include ethynyl, propynyl, butynyl, pentynyl and hexynyl.
  • the C 1-6 alkynyl group is preferably a C 1-4 alkynyl group, and more preferably a C 1-3 alkynyl group.
  • the C 1-6 alkyloxy group is an alkyloxy group having one to six carbon atom(s).
  • Examples of the C 1-4 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 and 2-ethylbutyloxy.
  • the C 1-6 alkyloxy group is preferably a C 1-4 alkyloxy group, and more preferably a C 1 alkyloxy group.
  • Examples of the C 6-14 aromatic hydrocarbon group include phenyl, naphthyl, and anthracenyl.
  • the C 6-14 aromatic hydrocarbon group is preferably phenyl or naphthyl, and more preferably phenyl.
  • the C 1-6 alkyl-carbonyl (acyl) group is a carbonyl group having the C 1-6 alkyl group as described above.
  • Examples of the C 1-6 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 and 2-ethylbutylcarbonyl.
  • the C 1-6 alkylcarbonyl group is preferably a
  • substituents may be further substituted with other substituents.
  • substituents include hydroxy groups, carboxy groups, nitro groups, amino groups and cyano groups.
  • Examples of the derivative of saccharide substituted with the substituent include cyclic polysaccharides (e.g., cyclodextrin such as ⁇ -cyclodextrin, O-cyclodextrin, ⁇ -cyclodextrin and the like), and derivatives of the linear polymer polysaccharide (e.g., cellulose).
  • Examples of the derivative of the cyclic polysaccharide include cyclic polysaccharides substituted with a substitutable alkyl group (e.g., methyl- ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin).
  • Examples of the derivative of the linear polymer polysaccharide includes a cellulose derivative.
  • the cellulose derivative is a cellulose derivative in which a hydrogen atom of at least one hydroxy group of the cellulose is substituted with a hydrophilic group.
  • the hydrophilic group in the cellulose derivative include carboxyalkyl (e.g., carboxy C 1-6 alkyl) and hydroxyalkyl (e.g., hydroxy C 1-6 alkyl).
  • the hydrophilic group in the cellulose derivative is preferably carboxyalkyl or hydroxyalkyl.
  • carboxyalkyl examples include carboxymethyl, carboxyethyl (1-carboxyethyl and 2-carboxyethyl), carboxypropyl (1-carboxypropyl, 2-carboxypropyl and 3-carboxypropyl), carboxyisopropyl (1-carboxy-2-methylethyl) and 2-carboxy-2-methylethyl), carboxybutyl (1-carboxybutyl, 2-carboxybutyl, 3-carboxybutyl and 4-carboxybutyl), carboxy t-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl, 3-carboxypentyl, 4-carboxypentyl and 5-carboxypentyl), carboxyhexyl (1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl, 5-carboxyhexyl and 6-carbox
  • hydroxyalkyl examples include hydroxymethyl, hydroxyethyl (1-hydroxyethyl and 2-hydroxyethyl), hydroxypropyl (1-hydroxypropyl, 2-hydroxypropyl and 3-hydroxypropyl), hydroxyisopropyl (1-hydroxy-2-methylethyl and 2-hydroxy-2-methylethyl), hydroxybutyl (1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl and 4-hydroxybutyl), hydroxy t-butyl, hydroxypentyl (1-hydroxypentyl, 2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl and 5-hydroxypentyl) and hydroxyhexyl (1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl, 5-hydroxyhexyl and 6-hydroxyhexyl).
  • cellulose derivative examples include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose or hydroxypropylmethyl cellulose.
  • the cellulose derivative also includes a nanocellulose derivative.
  • the nanocellulose derivative is a derivative of the nanocellulose described below.
  • the nanocellulose is a fibrous cellulose having a fiber width in a nanometer order.
  • the fiber width of the nanocellulose is, for example, 500 nm or less, and may be preferably 200 nm or less, more preferably 100 nm or less, still more preferably 50 nm or less, still further more preferably 10 nm or less, and particularly preferably 5 nm or less.
  • Examples of the cellulose derivatives also include salts thereof.
  • Examples of the salt includes salts of metal (e.g., a monovalent metal such as lithium, sodium, potassium, rubidium and cesium; and a bivalent metal such as calcium, magnesium and zinc), and salts of inorganic base (e.g., ammonia).
  • metal e.g., a monovalent metal such as lithium, sodium, potassium, rubidium and cesium; and a bivalent metal such as calcium, magnesium and zinc
  • salts of inorganic base e.g., ammonia
  • saccharide alcohol examples include: the monomer in which the carbonyl group is reduced in the monosaccharide as described above; a multimer in which the monomers are linked together, for example, by glycosidic bonds; and a multimer in which the monomer and the monosaccharide as constituent units are linked together, for example, by glycosidic bonds.
  • Examples of the monomer include tritols (e.g., glycerin), tetritols (e.g., erythritol and threitol), pentitols (e.g., xylitol, arabinitol and ribitol), hexitols (e.g., sorbitol, mannitol, iditol and galactitol), heptitols (e.g., volemitol and perseitol), and octyltols (e.g., erythrogalactooctytol).
  • tritols e.g., glycerin
  • tetritols e.g., erythritol and threitol
  • pentitols e.g., xylitol, arabinitol and ribitol
  • saccharide alcohol examples include: monomers such as xylitol, sorbitol, mannitol, galactitol, fucitol, volemitol, arabinitol, glycerin, iditol, erythritol, threitol, ribitol and the like; and dimers such as lactitol and maltitol.
  • a mixture of two or more (e.g., two, three, four and five) kinds of saccharides may be mixed with the extracellular vesicle-containing sample.
  • the present invention may include adding the saccharide to the extracellular vesicle-containing sample before mixing the extracellular vesicle-containing sample with the saccharide.
  • the saccharide may be added simultaneously with or separately from the chelating agent.
  • the concentration of the saccharide in the mixing is not particularly limited as long as the extracellular vesicle(s) can be stabilized.
  • concentration varies depending on factors such as kinds of the saccharides, and may be, for example, 2.5 mg/ml or more, preferably 3.0 mg/mL or more, more preferably 3.5 mg/mL or more, still more preferably 4.0 mg/mL or more, still further more preferably 4.5 mg/mL or more, and 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 varies depending on factors such as the kinds of saccharides, and may be, for example, 600 mg/mL or less, 400 mg/mL or less, 200 mg/mL or less, or 100 mg/mL or less, and may be preferably 95 mg/mL or less, more preferably 90 mg/mL or less, still more preferably 85 mg/mL or less, still further more preferably 80 mg/mL or less, and particularly preferably 75 mg/mL or less, 70 mg/mL or less, 60 mg/mL or less, or 50 mg/mL or less.
  • the concentration of the saccharide varies depending on factors such as the kinds of saccharides, and may be, for example, 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, and may be preferably 3.0 to 95 mg/mL, more preferably 3.5 to 90 mg/mL, still more preferably 4.0 to 85 mg/mL, still further more preferably 4.5 to 80 mg/mL, and 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.
  • the chelating agent is a compound having a coordination moiety capable of coordinating with a metal ion, or a salt thereof.
  • the number of the coordination moiety(ies) is preferably 2 or more, more preferably 3 or more (e.g., 3 or 6).
  • the coordination atom as the coordination moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom and a chlorine atom.
  • the coordination atom is preferably the oxygen atom or the phosphorus atom, and more preferably the oxygen atom.
  • Examples of a coordination group as the coordination moiety include a group having the abovementioned coordination atom.
  • the coordination group is preferably a carboxylic acid group or a phosphoric acid group, and more preferably the carboxylic acid group.
  • chelating agent examples include ethylenediaminetetraacetic acid (EDTA), glycoletherdiaminetetraacetic acid (EGTA), hydroxyethyl ethylenediaminetriacetic acid (HEDTA), hydroxyethyl iminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediaminetetra(methylene phosphonic acid) (EDTMP) and salts thereof.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA glycoletherdiaminetetraacetic acid
  • HEDTA hydroxyethyl ethylenediaminetriacetic acid
  • HIDA hydroxyethyl iminodiacetic acid
  • NTA nitrilotriacetic acid
  • EDTMP oxalic acid
  • ETMP ethylenediaminetetra(methylene phosphonic acid)
  • the salts include metal salts (e.g., monovalent metal salts such as sodium salts, potassium salts, and bivalent metal salts such as calcium salts, magnesium salts), 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 an inorganic acid such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid and the like, and salts with an organic acid such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, trifluoroacetic acid and the like).
  • metal salts e.g., monovalent metal salts such as sodium salts, potassium salts, and bivalent metal salts such as calcium salts, magnesium salts
  • a mixture of 2 or more (e.g., 2, 3, 4, or 5) kinds of chelating agents may be mixed with the extracellular vesicle-containing sample.
  • the present invention may include adding the chelating agent to the extracellular vesicle-containing sample before mixing the extracellular vesicle-containing sample with the chelating agent.
  • a concentration of the chelating agent in the mixing is not particularly limited as long as the extracellular vesicle(s) can be stabilized.
  • concentration varies depending on factors such as kinds of the chelating agents, and may be, for example, 1 mM or more, preferably 5 mM or more, more preferably 10 mM or more, still more preferably 15 mM or more, still further more preferably 20 mM or more, and particularly preferably 30 mM or more, 40 mM or more, or 50 mM or more.
  • such a concentration varies depending on factors such as the kinds of chelating agents, and may be, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, still more preferably 160 mM or less, still further more preferably 150 mM or less, and particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less.
  • 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, still more preferably 15 to 160 mM, still further more preferably 20 to 150 mM, and particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.
  • the extracellular vesicle-containing sample may be mixed with both of the saccharide and the chelating agent.
  • concentrations of the saccharide and the chelating agent to be used in the mixing can also be defined by their ratio.
  • concentration of the chelating agent per 10 mg/mL of the saccharide in the mixing varies depending on factors such as the kinds of saccharide and chelating agent, and may be, for example, 1 mM or more, preferably 5 mM or more, preferably 10 mM or more, still more preferably 15 mM or more, still further more preferably 20 mM or more, and particularly preferably 30 mM or more, 40 mM or more, or 50 mM or more.
  • such a concentration varies depending on factors such as the kinds of saccharide and chelating agent, and may be, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, still more preferably 160 mM or less, still further more preferably 150 mM or less, and particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less.
  • 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, still more preferably 15 to 160 mM, still further more preferably 20 to 150 mM, and particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.
  • the mixing can be carried out in any way.
  • the mixing can be carried out simultaneously or separately.
  • the extracellular vesicle-containing sample may be (1) mixed with the saccharide and the chelating agent simultaneously, (2) mixed with the chelating agent after being mixed with the saccharide, or (3) mixed with the saccharide after being mixed with the chelating agent.
  • the extracellular vesicle-containing sample may be mixed with the saccharide and the chelating agent simultaneously.
  • a mixing temperature may be, for example, 4 to 37° C., and preferably 15 to 30° C.
  • a mixing time is not particularly limited as long as the extracellular vesicle(s) can be stabilized, and can be controlled as appropriate.
  • the extracellular vesicle-containing sample may be left to stand after being mixed with a certain component such as the saccharide.
  • Whether the extracellular vesicle is stabilized by the mixing with the certain component such as the saccharide can be assessed, for example, by: comparing an index value that is measured after the mixing the extracellular vesicle with the certain component such as the saccharide, with a control value measured (under the same condition other than the presence or absence of the certain component such as the saccharide) for the extracellular vesicle in absence of the certain component such as the saccharide; and determining whether the index value is superior to the control value.
  • stability of a freeze-dried product may be assessed by: freeze-drying with a freeze dryer (manufactured by Tokyo Rikakikai Co., Ltd.) by setting a program at ⁇ 28° C.
  • index value it is possible to utilize an amount of an extracellular vesicle-marker and a measurement value of the number of particles corresponding to the extracellular vesicle.
  • the amount of the extracellular vesicle-marker can be measured by any well-known method in the relevant field.
  • examples of the measurement method include immunoassay and mass spectrometry.
  • examples of the immunoassay include a direct competitive method, an indirect competitive method and a sandwich method.
  • examples of such an immunoassay include chemiluminescent immunoassay (CLIA) [e.g., a chemiluminescent enzyme immunoassay (CLEIA)], turbidimetric immunoassay (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), and immunochromatography, Western blotting, immunostaining and fluorescence activated cell sorting (FACS).
  • proteomic analysis may be performed.
  • the extracellular vesicle-marker is a nucleic acid
  • examples of the measurement method include a hybridization method using a probe, a gene amplification method using a primer(s) (e.g., 2, 3 or 4 primers) and mass spectrometry.
  • the extracellular vesicle-marker is a component other than the protein and the nucleic acid
  • examples of the measurement method include immunoassays and mass spectrometric method.
  • metabolome analysis may be performed.
  • the number of particles corresponding to the extracellular vesicle(s) can be measured, for example, with an instrument such as a particle analysis instrument, an electron microscope, a flow cytometer and the like.
  • an instrument such as a particle analysis instrument, an electron microscope, a flow cytometer and the like.
  • a measurement of particle counts can be performed by NanoSight LM10 (manufactured by Malvern Instruments Ltd.).
  • NanoSight LM10 manufactured by Malvern Instruments Ltd.
  • the method of the present invention may further include freezing the mixture.
  • the present invention further provides the method of stabilizing the extracellular vesicle.
  • the method further includes:
  • the present invention further provides the method of stabilizing the extracellular vesicle.
  • the method further includes:
  • the present invention further provides the method of stabilizing the extracellular vesicle.
  • the method further includes:
  • the freezing may be either the freeze-drying or a freezing of solution (non-freeze-drying).
  • the freeze-drying is preferred in the case of preserving the extracellular vesicle(s) for a longer period.
  • the present invention also provides an extracellular vesicle-stabilizing reagent.
  • the reagent of the present invention contains the saccharide or the chelating agent.
  • the reagents of the present invention may contain the saccharide and the chelating agent.
  • the saccharide and the chelating agent are the same as those described above.
  • the reagent of the present invention may include another component (e.g., another component useful in the stabilization of extracellular vesicle(s)), in addition to the saccharide and chelating agents.
  • the reagent of the present invention is an extracellular vesicle-stabilizing composition containing the saccharide.
  • the reagent of the present invention is an extracellular vesicle-stabilizing composition containing a chelating agent.
  • the composition of the present invention is an extracellular vesicle-stabilizing composition containing both the saccharide and the chelating agent.
  • the reagent of the present invention can be mixed with the extracellular vesicle-containing sample for use.
  • the mixture of the extracellular vesicle-containing sample and the reagent of the present invention may be frozen.
  • the reagent of the present invention can be used as an extracellular vesicle-cryopreservation stabilizing reagent.
  • the freezing can be either the freeze-drying or the freezing of solution (non-freeze-drying). The freeze-drying is preferred in the case of preserving the extracellular vesicle(s) for a longer period.
  • the concentration of the saccharide in the composition is not particularly limited, as long as the extracellular vesicle(s) can be stabilized after the mixing with the extracellular vesicle-containing sample.
  • concentration varies depending on factors such as the kind of the saccharide, and may be, for example, 2.7 mg/mL or more, preferably 3.0 mg/mL or more, more preferably 3.5 mg/mL or more, still more preferably 4.0 mg/mL or more, still further more preferably 4.5 mg/mL or more, and 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 varies depending on factors such as the kind of saccharide, and may be, for example, 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, still more preferably 85 mg/mL or less, still further more preferably 80 mg/mL, particularly preferably 75 mg/mL or less, 70 mg/mL or less, 60 mg/mL or less, or 50 mg/mL or less.
  • the concentration of the saccharide varies depending on factors such as the kind of saccharide, and may be, for example, 2.7 to 800 mg/mL, 2.7 to 600 mg/mL, 2.7 to 400 mg/mL, 2.7 to 200 mg/mL or 2.7 to 100 mg/mL, preferably 3.0 to 95 mg/mL, more preferably 3.5 to 90 mg/mL, still more preferably 4.0 to 85 mg/mL, still further more preferably 4.5 to 80 mg/mL, and 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.
  • the concentration of the chelating agent in the composition is not particularly limited, as long as the extracellular vesicle(s) can be stabilized after the mixing with the extracellular vesicle-containing sample.
  • concentration varies depending on factors such as the kind of the chelating agent, and may be, for example, 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, still more preferably 30 mM or more, still further more preferably 40 mM or more, and particularly preferably 50 mM or more.
  • such a concentration varies depending on factors such as the kind of chelating agents, and may be, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, still more preferably 160 mM or less, still further more preferably 150 mM or less, and particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less.
  • 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, still more preferably 15 to 160 mM, still further more preferably 20 to 150 mM, and particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.
  • the concentrations of the saccharide and the chelating agent can also be defined by their ratio.
  • the concentration of the chelating agent per 10 mg/mL of the saccharide varies depending on factors such as the kinds of saccharide and chelating agent, and may be, 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, still more preferably 30 mM or more, still further more preferably 40 mM or more, and particularly preferably 50 mM or more.
  • such a concentration varies depending on factors such as the kinds of saccharide and chelating agents, and may be, for example, 200 mM or less, preferably 180 mM or less, more preferably 170 mM or less, still more preferably 160 mM or less, still further more preferably 150 mM or less, and particularly preferably 140 mM or less, 120 mM or less, or 100 mM or less.
  • 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, still more preferably 15 to 160 mM, still further more preferably 20 to 150 mM, and particularly preferably 30 to 140 mM, 40 to 120 mM, or 50 to 100 mM.
  • the composition of the present invention is dissolved in an aqueous solution for use.
  • the composition of the present invention may be either a non-aqueous composition (e.g., mixture powder) containing the saccharide and/or the chelating agent or an aqueous solution containing the saccharide and/or the chelating agent. From the viewpoint of quick and simple use and so on, the aqueous solution containing the saccharide and/or the chelating agent is preferred.
  • the aqueous solution include water (e.g., distilled water, sterilized water, sterilized distilled water and pure water) and buffer. The buffer is preferred.
  • buffer examples 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, borate buffer, 3-morpholinoproponesulfonic 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-piperazinylethanesulfonic acid (HEPES) buffer.
  • MES 2-morpholinoethanesulfonic acid
  • Tris tris(hydroxymethyl)aminomethane
  • MOPS 3-morpholinoproponesulfonic acid
  • the buffer has neutral pH. More specifically, such a pH is preferably 5.0 or more, more preferably 5.5 or more, and still more preferably 6.0 or more. Also, the pH is preferably 9.0 or less, more preferably 8.5 or less, and still more preferably 8.0 or less.
  • the pH can be measured by well-known methods in the relevant field. Preferably, it is possible to employ a value measured at 25° C. with a pH meter having a glass electrode, as the pH.
  • composition of the present invention can be used by being mixed with the extracellular vesicle-containing sample as appropriate.
  • a mixing ratio of the composition of the present invention and the extracellular vesicle-containing sample (the extracellular vesicle-containing sample/the composition) is, for example, 1/30 to 30, preferably 1/20 to 20, more preferably 1/10 to 10, and still more preferably 1/10 to 1.
  • a volume of the aqueous solution is, for example, 1 ⁇ L to 100 mL.
  • the volume of the aqueous solution may be 10 ⁇ L or more, 100 ⁇ L or more, or 1000 ⁇ L or more.
  • the volume of the aqueous solution may be 50 mL or less, 10 mL or less, or 2 mL or less.
  • the reagent of the present invention is a kit including the saccharide and/or the chelating agent.
  • the saccharide and/or the chelating agent can be provided in a solid form or in an aqueous solution form, and is preferably provided in the aqueous solution form. Therefore, the kit of the present invention may be provided in an aqueous solution form containing the saccharide, may be provided in an aqueous solution form containing the chelating agent, may be provided in an aqueous solution form containing both the saccharide and the chelating agent, or may be provided in a first aqueous solution form containing the saccharide and a second aqueous solution form containing the chelating agent.
  • the aqueous solution is described above, and preferably the buffer. Examples and pH of the buffer are described above.
  • the abovementioned concentrations of saccharide and chelating agent in the composition of the present invention can be applied also to a concentration of the saccharide in the first aqueous solution, a concentration of the chelating agent in the second aqueous solution and a concentration ratio of the saccharide in the first aqueous solution and the chelating agent in the second aqueous solution.
  • the abovementioned mixing ratios and the volumes in the composition of the present invention can be applied also to a mixing ratio of the first and second aqueous solutions and the extracellular vesicle-containing sample and volumes of the first and second aqueous solutions.
  • the present invention also provides a mixture containing the saccharide and/or the chelating agent, and the extracellular vesicle.
  • the mixture of the present invention may further include the aqueous solution (e.g., the buffer solution described above).
  • the form of the mixture is not particularly limited, and preferably the aqueous solution or a frozen product thereof (e.g., a freeze-dried product).
  • the mixture of the present invention can be obtained by treating the extracellular vesicle-containing sample in the method of the present invention or with the reagent thereof as described above.
  • the mixture of the present invention is useful in preservation of the extracellular vesicle(s).
  • concentrations described above regarding the method of the present invention can be applied also to a concentration of the saccharide or the chelating agent in the mixture, and a concentration ratio of the saccharide and the chelating agent.
  • concentration (particle counts/mL) of extracellular vesicle(s) in the mixture is, for example, 1 ⁇ 10 2 to 1 ⁇ 10 15 , preferably 1 ⁇ 10 3 to 1 ⁇ 10 14 , more preferably 1 ⁇ 10 4 to 1 ⁇ 10 13 , still more preferably 1 ⁇ 10 5 to 1 ⁇ 10 12 , and particularly preferably 1 ⁇ 10 6 to 1 ⁇ 10 11 .
  • volume of the aqueous solution is, for example, 1 ⁇ L to 100 mL.
  • the volume of aqueous solution may be 10 ⁇ L or more, 100 ⁇ L or more, or 1000 ⁇ L or more.
  • the volume of aqueous solution may be 50 mL or less, 10 mL or less, or 2 mL or less.
  • a culture supernatant of human non-small cell lung carcinoma cell line H1299 cultured in serum-free medium for three days was used as a sample.
  • the culture supernatant was centrifuged at 2,000 ⁇ g at 4° C. for 5 minutes, then filtered through a 0.22 ⁇ m filter (manufactured by Millipore Corp.), and then concentrated using Amicon Ultra-15 (manufactured by Millipore Corp.).
  • the concentrate was centrifuged at 20,000 ⁇ g at 4° C. for 15 minutes. Then, the supernatant was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour.
  • Protein quantification was performed for the recovered exosomes with Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.). Then, the recovery buffer was further added to each of the recovered exosomes so as to prepare solutions containing the same concentration of protein. Next, 1 volume of the recovered exosomes was mixed with 24 volumes of the freeze-drying buffer. After dispensation, the solution was freeze-dried with a freeze dryer (manufactured by Tokyo Rikakikai Co., Ltd.) with a program of ⁇ 28° C. for 2 hours, ⁇ 10° C. for 4 hours and subsequent 20° C., in order to obtain a freeze-dried product containing freeze-dried exosomes. As the freeze-drying buffer, PBS or EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) was used. pH of PBS used was 7.4 (same in Examples 1 to 3).
  • freeze-dried product was dissolved in milli-Q water (manufactured by Millipore Corp.) or 50 mM EDTA/50 mM EGTA/H 2 O (dissolving buffer).
  • the amount of the dissolved freeze-dried exosomes was measured by ELISA system. Specifically, PBS (pH 7.4) containing anti-CD9 antibody prepared in the applicant was added to a 96-well ELISA plate (manufactured by NUNC Inc.), and incubated overnight at 4° C. Then, each well was washed three times with PBS containing 0.05 wt % of Tween (registered trademark) 20 (PBS-T), 200 ⁇ L of PBS containing 0.5 wt % Casein was added, and incubated for 2 hours at room temperature.
  • PBS pH 7.4
  • Tween registered trademark
  • Lumipulse (registered trademark) substrate solution manufactured by Fujirebio Inc.
  • emission count was measured at a 477 nm wavelength. The count of the sample obtained without freeze-drying is defined as 100%.
  • the chelating agent can stabilize the exosomes in the freeze-drying of exosomes.
  • a culture supernatant of human colon carcinoma cell line SW480 cultured in the serum-free medium for three days was used as a sample.
  • the culture supernatant was centrifuged at 2,000 ⁇ g at 4° C. for 5 minutes, then filtered through the 0.22 ⁇ m filter (manufactured by Millipore Corp.), and then concentrated using Amicon Ultra-15 (manufactured by Millipore Corp.).
  • the concentrate was centrifuged at 20,000 ⁇ g at 4° C. for 15 minutes.
  • the supernatant was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour.
  • the supernatant was discarded, and then PBS was added to resuspend the precipitate.
  • the resuspension was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour.
  • the supernatant was discarded, and PBS was newly added to resuspend the precipitate in order to recover exosomes.
  • Protein quantification was performed for the recovered exosomes with Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.). Then, PBS was further added to each of the recovered exosomes to prepare solutions containing the same concentration of protein. Next, 1 volume of the recovered exosomes was mixed with 24 volumes of the freeze-drying buffer. After dispensation, the solution was freeze-dried with the above-mentioned freeze dryer with a program of ⁇ 28° C. for 2 hours, ⁇ 10° C. for 4 hours and subsequent 20° C., in order to obtain a freeze-dried product containing freeze-dried exosomes. As the freeze-drying buffer, PBS, EDTA/EGTA/PBS (final conc.
  • sucrose/PBS final conc. 10 mg/mL sucrose
  • EDTA/EGTA/sucrose/PBS final conc. 50 mM EDTA/50 mM EGTA/10 mg/mL sucrose
  • the amount of the dissolved freeze-dried exosomes was measured by ELISA system. Specifically, PBS (pH 7.4) containing anti-CD9 antibody prepared in the applicant was added to the 96-well ELISA plate (manufactured by NUNC Inc.), and incubated overnight at 4° C. Then, each well was washed three times with PBS-T, 200 ⁇ L of PBS containing 0.5 wt % Casein was added, and incubated for 2 hours at room temperature. After washing with PBS-T, 100 ⁇ L of the dissolved freeze-dried product diluted with PBS was added to each well and incubated for 1 hour at 37° C.
  • the exosome amount was increased ( FIG. 2 ). Also, in the freeze-drying buffer containing the sucrose and the chelating agent, the exosome amount was further increased.
  • the saccharide can stabilize the exosomes in the freeze-drying of exosomes.
  • the stability of the exosomes with the saccharide can be enhanced in the presence of the chelating agent.
  • the stability was assessed on the basis of particle counts of the freeze-dried exosomes by measurement using NanoSight for a freeze-dried sample.
  • a culture supernatant of human colon carcinoma cell line SW1116 cultured in the serum-free medium for three days was used as a sample.
  • the culture supernatant was centrifuged at 2,000 ⁇ g at 4° C. for 5 minutes, then filtered through the 0.22 ⁇ m filter (manufactured by Millipore Corp.), and then concentrated using Amicon Ultra-15 (manufactured by Millipore Corp.). The concentrate was centrifuged at 20,000 ⁇ g at 4° C. for 15 minutes. Next, the supernatant was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour.
  • Protein quantification was performed for the recovered exosomes with Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.). Then, the recovery buffer was further added to each of the recovered exosomes to prepare solutions containing the same concentration of protein. Next, 1 volume of the recovered exosomes was mixed with 24 volumes of the freeze-drying buffer. After dispensation, the solution was freeze-dried with the abovementioned freeze dryer with a program of ⁇ 28° C. for 2 hours, ⁇ 10° C. for 4 hours and subsequent 20° C., in order to obtain a freeze-dried product containing freeze-dried exosomes. As the freeze-drying buffer, PBS and EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) (ED/EG) was used.
  • the number and size distribution of the particles were measured by NanoSight LM10 (manufactured by Malvern Instruments) for the dissolved freeze-dried exosomes. The measurement was performed for 30 seconds and repeated three times. The analysis was carried out with a detection threshold of 15.
  • the chelating agent can stabilize the exosomes in the freeze-drying of exosomes also by the measurement of the number of particle.
  • EDTA-2Na disodium ethylenediaminetetraacetate
  • EGTA glycoletherdiaminetetraacetic acid
  • HEDTA hydroxyethyl ethylenediaminetriacetic acid
  • HIDA hydroxyethyliminodiacetic acid
  • NTA nitrilotriacetic acid
  • EDTMP ethylenediaminetetra (methylenephosphonic acid)
  • HEDTA, HIDA, NTA, EDTMP were from Tokyo Chemical Industry Co., Ltd.
  • oxalic acid dihydrate was from Wako Pure Chemical Industries, Ltd.
  • 300 ⁇ L of a serum specimen or a plasma specimen was diluted with 600 ⁇ L of PBS or each chelating agent-added PBS.
  • concentrations of each chelating agent to be used were 1, 10, and 50 mM at final concentrations (provided, 1, 10, and 30 mM only for EDTMP).
  • the solutions were left to stand at room temperature for 30 minutes, and then centrifuged at 20,000 ⁇ g at 4° C. for 15 minutes. The resulting supernatant was transferred to a new tube, and magnetic beads (Protein G Dynabeads) immobilized with 2 ⁇ g of the monoclonal antibody recognizing CD9 prepared in the applicant were added thereto. After reacting at 4° C.
  • the culture supernatant of the human non-small cell lung carcinoma cell line H1299 cultured in the serum-free medium for three days was used as a sample.
  • the exosomes were recovered by the recovery method in Example 2.
  • Protein quantification was performed for the recovered exosomes with Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.). Then, PBS was further added to each of the recovered exosomes to prepare solutions containing the same concentration of protein. Next, 1 volume of the recovered exosomes was mixed with 9 volumes of the freeze-drying buffer. After dispensation, the solution was freeze-dried with a freeze dryer (manufactured by Genevac Ltd.) in order to obtain a freeze-dried product containing freeze-dried exosomes.
  • PBS and PBS solutions each of that contains one of lactose, fructose, methyl ⁇ -cyclodextrin (MPCD), hydroxypropyl ⁇ -cyclodextrin (Hp ⁇ CD), ⁇ -cyclodextrin ( ⁇ CD), glucose, lactitol, sorbitol, xylitol, sucrose, trehalose, mannitol and xylose in a final concentration of 2.0 mg/mL, 10 mg/mL and 50 mg/mL, were used.
  • MPCD methyl ⁇ -cyclodextrin
  • Hp ⁇ CD hydroxypropyl ⁇ -cyclodextrin
  • ⁇ CD ⁇ -cyclodextrin
  • the amount of the dissolved freeze-dried exosomes was measured by ELISA system. Specifically, PBS (pH 7.4) containing anti-CD9 antibody prepared in the applicant was added to the 96-well ELISA plate (manufactured by NUNC Inc.), and incubated overnight at 4° C. Then, each well was washed three times with PBS-T, 200 ⁇ L of PBS containing 0.5 wt % BSA was added, and incubated for 2 hours at room temperature. After washing with PBS-T, 100 ⁇ L of the dissolved freeze-dried product was added to each well and incubated for 1 hour at 37° C.
  • each of the saccharides can stabilize the exosomes in the freeze-drying of exosomes, and in particular, exhibit a high stabilization effect in the case of 10 to 50 mg/mL of the saccharide.
  • PBS EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) (ED/EG), or either one thereof that contains lactose, ⁇ -cyclodextrin ( ⁇ CD), glucose or lactitol in a final concentration of 10 mg/mL, was used as the freeze-drying buffer.
  • EDTA/EGTA/PBS final conc. 50 mM EDTA/50 mM EGTA
  • ⁇ CD ⁇ -cyclodextrin
  • glucose or lactitol in a final concentration of 10 mg/mL
  • Human serum was used as a sample.
  • the serum was centrifuged at 2,000 ⁇ g at 4° C. for 5 minutes, and then centrifuged at 20,000 ⁇ g at 4° C. for 15 minutes.
  • the supernatant was centrifuged at 100,000 ⁇ g at 4° C. for 3 hours.
  • the supernatant was discarded, and then PBS was added to resuspend the precipitate.
  • the resuspension was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour. The supernatant was discarded, and PBS was newly added to resuspend the precipitate in order to recover exosomes.
  • PBS EDTA/EGTA/PBS (final conc. 50 mM EDTA/50 mM EGTA) (ED/EG), or either one thereof that contains lactose, glucose or lactitol in a final concentration of 10 mg/mL or 50 mg/mL, was used as the freeze-drying buffer.
  • the exosome amount was increased ( FIG. 7 ).
  • the exosome amount was further increased in the case of using the freeze-drying buffers containing both of the saccharide and the chelating agent.
  • CMC carboxymethyl cellulose
  • the culture supernatant of the human non-small cell lung carcinoma cell line H1299 cultured in the serum-free medium for three days was used as a sample.
  • the culture supernatant was centrifuged at 2,000 ⁇ g at 4° C. for 5 minutes, then filtered through the 0.22 ⁇ m filter (manufactured by Millipore Corp.), and then concentrated using Amicon Ultra-15 (manufactured by Millipore Corp.). The concentrate was centrifuged at 100,000 ⁇ g at 4° C. for 3 hours. Next, the supernatant was discarded, and then PBS was added to resuspend the precipitate. Then, the resuspension was centrifuged at 100,000 ⁇ g at 4° C. for 1 hour. The supernatant was discarded, and PBS was newly added in order to recover exosomes.
  • Protein quantification was performed for the recovered exosomes with Qubit (trademark) Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.), and concentration was adjusted. Next, 1 volume of the recovered exosomes was mixed with an equivalent volume of the freeze-drying buffer, frozen at ⁇ 20° C. for 3 hours, and then dried with a centrifugal evaporator (manufactured by Scrum Inc.). Then, the freeze-dried product was dissolved in milli-Q water (manufactured by Millipore Corp.) and diluted with PBS. PBS and CMC/PBS (final conc. 0.2 wt % or 1 wt % CMC) were used as the freeze-drying buffers.
  • the amount of the dissolved freeze-dried exosomes was evaluated by ELISA system in which the anti-CD9 antibody prepared in the applicant was used in a solid phase and the biotinylated anti-CD9 antibody prepared in the applicant and SA-ALP (manufactured by GeneTex, Inc.) were used for detection. In the evaluation, the count of the sample obtained without the freeze-drying was defined as 100%.
  • CMC can stabilize the exosomes in the freeze-drying of exosomes.

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