Method for separating extracellular vesicles and carrier used therefor

Abstract

A method for separating extracellular vesicles comprises: bringing a sample containing extracellular vesicles into contact with a carrier comprising a ligand for capturing extracellular vesicles; and bringing the carrier after being brought into contact with the sample into contact with a buffer containing a saccharide, wherein the saccharide is at least one saccharide selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, and the ligand is capable of binding to the saccharide.

Description

Method for separating extracellular vesicles and carriers used therefor

[0001] The present invention relates to a method for separating extracellular vesicles and a carrier used therefor.

[0002] Extracellular vesicles (EVs) secreted from cells are granules with a diameter of approximately 100 to 1000 nm surrounded by a lipid bilayer membrane. EVs have various membrane proteins in their membranes, and contain various proteins such as cytokines and microRNAs inside. In recent years, attention has been focused on the function of EVs as a mediator of intercellular communication in the body, as well as the relationship between EVs and in vivo processes such as immune responses and diseases such as cancer. Research is also being conducted on analytical, diagnostic, and therapeutic methods using EVs.

[0003] To study EVs, they must first be isolated from biological samples. Conventional methods for obtaining EVs include ultracentrifugation of a sample containing EVs and density fractionation of the resulting precipitate using a density gradient method, and affinity methods using antibodies against antigenic proteins on the surface of EVs (Non-Patent Document 1). However, the former method requires expensive equipment for ultracentrifugation and makes it difficult to separate EVs from substances of the same density. The latter method requires the use of surfactants or acidic buffers to elute EVs from the antibodies, making it difficult to obtain EVs while maintaining their original functions. An affinity method using a protein that binds to phosphatidylserine on the surface of EVs is known, and EVs are obtained using a calcium ion chelator (Patent Document 1). Patent Documents 2 to 4 disclose methods for capturing and isolating exosomes on a carrier equipped with lectins. However, neither of these documents examines whether the obtained EVs or exosomes have physiological activity in living organisms.

[0004] Patent Document 5 describes a method for producing magnetic particles that exhibit high sensitivity and low noise with little nonspecific adsorption of proteins, nucleic acids, etc., and that include the steps of: forming a hydrophobic first polymer layer on the surface of base particles containing superparamagnetic microparticles; forming a second polymer layer having glycidyl groups on at least the surface of the first polymer layer; and chemically modifying the glycidyl groups to introduce polar groups containing one or more atoms of at least one type selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms.

[0005] Japanese Patent No. 6824742 International Publication No. 2010 / 141862 Japanese Patent Application Laid-Open No. 2021-115528 Japanese Patent Application Laid-Open No. 2023-503713 Japanese Patent Application Laid-Open No. 2008-032411

[0006] Journal of Extracellular Vesicles, 2013, May, 27; 2, doi:10.3402

[0007] The present invention provides a method for more efficiently separating and obtaining extracellular vesicles from biological samples.

[0008] The present invention provides the following as representative embodiments. [1] A method for separating extracellular vesicles, comprising: contacting a sample containing extracellular vesicles with a carrier having a ligand for capturing extracellular vesicles; and contacting the carrier, after contact with the sample, with a buffer containing a saccharide, wherein the saccharide is at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, and the ligand has a binding ability to the saccharide. [2] The method according to [1], wherein the saccharide is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine. [3] The method according to [1] or [2], wherein the ligand is a lectin. [4] The method according to [3], wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin. [5] The method according to any one of [1] to [4], further comprising washing the carrier after contact with the sample before contacting it with the saccharide-containing buffer. [6] The method according to any one of [1] to [5], further comprising passing the sample through an ultrafiltration membrane before contacting it with the carrier. [7] The method according to any one of [1] to [6], wherein the extracellular vesicles have tetraspanin on their surface. [8] The method according to [7], wherein the tetraspanin comprises at least one selected from the group consisting of CD9, CD63, and CD81. [9] The method according to any one of [1] to [8], wherein the carrier is a particulate carrier having a particle size of 1 to 60 μm.

[10] A carrier for separating extracellular vesicles, comprising: a carrier substrate; and a ligand immobilized on the carrier substrate, wherein the carrier is contacted with a sample containing extracellular vesicles, and then with a buffer containing a sugar to dissociate the extracellular vesicles captured on the carrier from the ligand, thereby separating the extracellular vesicles, wherein the sugar is at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, and the ligand has a binding ability to the sugar.

[11] The carrier according to

[10] , wherein the sugar is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine.

[12] The carrier according to

[10] or

[11] , wherein the ligand is a lectin.

[13] The carrier according to

[12] , wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin.

[14] The carrier according to any one of

[10] to

[13] , wherein the extracellular vesicles have tetraspanin on their surface.

[15] The carrier according to

[14] , wherein the tetraspanin comprises at least one selected from the group consisting of CD9, CD63, and CD81.

[16] The carrier according to any one of

[10] to

[15] , wherein the carrier is a particulate carrier having a particle size of 1 to 60 μm.

[17] A kit for separating extracellular vesicles, comprising: an extracellular vesicle separation carrier; and a buffer solution containing sugars. The extracellular vesicle separation carrier comprises: a carrier substrate; and a ligand immobilized on the carrier substrate, the ligand having a binding ability to the sugars. The buffer solution containing the sugars is used to dissociate the extracellular vesicles captured on the extracellular vesicle separation carrier from the ligand, and the sugar is at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom.

[18] The kit according to

[17] , wherein the saccharide is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine.

[19] The kit according to

[17] or

[18] , wherein the ligand is a lectin.

[20] The kit according to

[19] , wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin.

[21] The kit according to any one of

[17] to

[20] , wherein the extracellular vesicles have a tetraspanin on their surface.

[22] The kit according to

[21] , wherein the tetraspanin comprises at least one selected from the group consisting of CD9, CD63, and CD81.

[23] The kit according to any one of

[17] to

[22] , wherein the carrier for separating extracellular vesicles is a particulate carrier having a particle diameter of 1 to 60 μm.

[0009] According to the present invention, the efficiency of separating extracellular vesicles (EVs) from biological samples can be improved. The EV separation method of the present invention allows for the production of EVs in high yield, and is therefore useful for analyzing the structure of EVs and the substances contained within EVs. Furthermore, according to the present invention, it is possible to separate and obtain extracellular vesicles in a state in which they retain their original functions. The extracellular vesicles obtained according to the present invention are physiologically active in living organisms and can exert their functions in vivo, and are useful for the analysis of the functions of extracellular vesicles in vivo, the search for substances that regulate these functions, the development of pharmaceuticals, etc.

[0010] Schematic diagram showing an outline of the EV isolation method of the present invention. Relative expression level of TNF-α mRNA in macrophage-like cells to which an EV solution was added.

[0011] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0012] In this specification, the expression "A to B" or the like representing a range of numerical values ​​is synonymous with "greater than or equal to A and less than or equal to B," and A and B are included in the range of numerical values.

[0013] As used herein, the term "ligand" in connection with affinity separation refers to a molecule that binds to a target substance of affinity separation.

[0014] As used herein, "extracellular vesicles (EVs)" are small vesicular granules surrounded by a lipid bilayer membrane secreted by living cells. There are several types of EVs, differing in their origin, membrane structure (membrane proteins contained), internal contents, and vesicle size. EVs are primarily classified by their origin into exosomes derived from endosomes, microvesicles derived from the cell membrane, and apoptotic bodies released from cells undergoing apoptosis. Of these three, exosomes are relatively small (approximately 50-200 nm) and contain endosome-associated proteins in their membrane structure. For example, exosome membranes are rich in tetraspanins (e.g., CD9, CD63, and CD81), a family of transmembrane proteins. Plasma membrane-derived microvesicles are larger (approximately 100-1000 nm) and contain marker molecules such as RhoA and integrin family proteins. It is also known that some microvesicles contain tetraspanins (e.g., CD9 and CD81). Apoptotic bodies are fragments of cells that have undergone programmed cell death. Apoptotic bodies are generally approximately 500 to 5,000 nm in size and are characterized by exposing nuclear components and phosphatidylserine on their surface. Meanwhile, some small EVs released during apoptosis exhibit exosome-like properties, such as high CD63 expression. Academically, tetraspanins such as CD9, CD63, and CD81 are considered markers for EVs, but it is recognized that it is difficult to strictly distinguish between EV subtypes such as exosomes, microvesicles, and apoptotic bodies based solely on tetraspanin expression profiles.

[0015] [Carrier for separating extracellular vesicles (EVs)] In one embodiment, the present invention provides a carrier for separating EVs. Hereinafter, the carrier provided by the present invention is referred to as the "carrier for separating EVs of the present invention" or simply as the "carrier of the present invention." The carrier of the present invention is a carrier for affinity separation of EVs and comprises a ligand for capturing EVs.

[0016] (Ligand) The ligand provided in the carrier of the present invention is selected from those capable of binding to saccharides. Examples of saccharides to which the ligand can bind include at least one saccharide selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides composed of these saccharides, more preferably at least one saccharide selected from the group consisting of oligosaccharides and polysaccharides composed of glucose, glucosamine, or N-acetylglucosamine. Examples of oligosaccharides and polysaccharides include chitobiose, N,N'-diacetylchitobiose, oligoglucosamine (chitosan oligosaccharide), and oligo-N-acetylglucosamine (chitin oligosaccharide), and preferably N,N'-diacetylchitobiose and oligo-N-acetylglucosamine (chitin oligosaccharide), more preferably oligo-N-acetylglucosamine (chitin oligosaccharide). The ligand is preferably a saccharide-binding protein, more preferably a lectin. In this specification, an oligosaccharide refers to a sugar in which 2 to 9 monosaccharides, preferably 2 to 6 monosaccharides, are linked together, and a polysaccharide refers to a sugar in which 10 or more monosaccharides are linked together.

[0017] Preferably, the ligand is a lectin that specifically binds to one or more of the saccharides listed above. Examples of such lectins include wheat germ lectin (WGA), tomato lectin (LEL), potato lectin (STL), and Datura stramonium lectin (DSL), more preferably wheat germ lectin (WGA) and potato lectin (STL7), and particularly preferably wheat germ lectin (WGA). In a preferred embodiment, one or more of these lectins are immobilized as a ligand on the carrier of the present invention.

[0018] (Carrier substrate) In the carrier of the present invention, the ligand is immobilized on a solid-phase carrier substrate. Examples of materials for the carrier substrate include polymer compounds such as polystyrenes, polyethylenes, polypropylenes, polyesters, poly(meth)acrylonitriles, styrene-butadiene copolymers, poly(meth)acrylic acid esters, fluororesins, cross-linked dextran, and polysaccharides; glass; metal; latex; magnetic materials; resin compositions containing magnetic materials; and combinations thereof. The shape of the carrier substrate is not particularly limited, and examples include tray-shaped, spherical, particulate, fibrous, rod-shaped, disc-shaped, container-shaped, tubular, cell-shaped, and microplate-shaped.

[0019] In one embodiment, the carrier of the present invention is a particulate carrier comprising a particulate carrier substrate and the ligand fixed to the carrier substrate. Examples of the particulate carrier substrate include polymer porous particles and magnetic particles. Porous particles have a large surface area and can increase the amount of adsorption, while magnetic particles can improve handling.

[0020] (Porous Particles) The porous particles can be produced by preparing a mixed solution in which a monomer composition and a porosifying agent are suspended in an aqueous medium, adding a polymerization initiator thereto, and polymerizing the monomer under heating.

[0021] The monomer composition used to produce the porous particles preferably contains a functional group-containing monomer. The functional group-containing monomer is preferably a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group. Examples of such monomers include (meth)acrylate monomers having a cyclic ether group, aromatic vinyl monomers having a cyclic ether group, (meth)acrylate monomers having an isocyanate group, and unsaturated dicarboxylic acid anhydride monomers; (meth)acrylic acid, 3,4-epoxy-1-butene, and 3,4-epoxy-3-methyl-1-butene. Of these, (meth)acrylate monomers having a cyclic ether group are preferred, with glycidyl methacrylate being more preferred. These monomers can be used alone or in combination of two or more. The total content of the functional group-containing monomers in the monomer composition is preferably 35% by mass or more, more preferably 45% by mass or more, and even more preferably 55% by mass or more, and is preferably 99% by mass or less, more preferably 95% by mass or less, and even more preferably 90% by mass or less.

[0022] The monomer composition may further contain a monomer other than the functional group-containing monomer (hereinafter also referred to as "other monomer"). Examples of the other monomer include a polymerizable unsaturated group-containing monomer that does not have a functional group capable of immobilizing a ligand. The other monomer is roughly classified into a non-crosslinkable monomer and a crosslinkable monomer, and these may be used singly or in combination.

[0023] Preferred examples of the non-crosslinkable monomer include aromatic vinyl non-crosslinkable monomers. Examples of the aromatic vinyl non-crosslinkable monomer include styrenes such as styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, and 4-tert-butylstyrene; and vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene. These may be used alone or in combination of two or more. The total content of the non-crosslinkable monomers in the monomer composition is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.1% by mass or more, and is preferably 30% by mass or less, more preferably 15% by mass or less, and even more preferably 5% by mass or less.

[0024] The crosslinkable monomer is preferably a di- to penta-functional crosslinkable monomer, and more preferably a di- or tri-functional crosslinkable monomer. Preferred examples of the crosslinkable monomer include aromatic vinyl crosslinkable monomers. Examples of the aromatic vinyl crosslinkable monomer include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, and divinylnaphthalene. These may be used alone or in combination of two or more. The total content of the crosslinkable monomer in the monomer composition is preferably 1% by mass or more, more preferably 5% by mass or more, and even more preferably 10% by mass or more, and is preferably 50% by mass or less, more preferably 40% by mass or less, and even more preferably 30% by mass or less.

[0025] Examples of the porogen include ketones such as diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone, and cyclohexanone, and polysaccharides such as methyl cellulose and ethyl cellulose. These porogens can be used alone or in combination of two or more. The total amount of the porogen used in producing the porous particles is preferably 40 parts by mass or more, more preferably 70 parts by mass or more, per 100 parts by mass of the total amount of monomers, and is preferably 600 parts by mass or less, more preferably 400 parts by mass or less.

[0026] Examples of the aqueous medium used in producing the porous particles include aqueous solutions of water-soluble polymers. Examples of the water-soluble polymers include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinylpyrrolidone, starch, and gelatin. The total amount of the aqueous medium used in producing the porous particles is usually about 200 to 7,000 parts by mass per 100 parts by mass of the total amount of monomers.

[0027] The polymerization initiator used in the production of the porous particles is preferably a radical polymerization initiator, and examples thereof include azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, benzoyl peroxide-dimethylaniline, etc. The total amount of the polymerization initiator used in the production of the porous particles is usually about 0.01 parts by mass or more and 10 parts by mass or less per 100 parts by mass of the total amount of monomers.

[0028] In the production of the porous particles, the polymerization temperature of the monomer may be determined depending on the polymerization initiator, but is usually about 2 to 100° C., preferably 50 to 100° C. The polymerization time of the monomer is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

[0029] (Magnetic Particles) From the viewpoint of ease of solid-liquid separation and cleaning, the carrier substrate is preferably magnetic particles. The magnetic particles are preferably magnetic particles containing fine particles of a magnetic material in a resin. Examples of the magnetic material include iron oxide (FeO), ferrous oxide (γ-FeO), ferrite, iron, manganese, nickel, cobalt, chromium, and other metals; and alloys of cobalt, nickel, manganese, and other metals. Examples of the resin include hydrophobic polymers and hydrophilic polymers.

[0030] A preferred example of the magnetic particles is a magnetic particle comprising a base particle containing superparamagnetic particles and a polymer layer formed on the surface thereof. An example of such a magnetic particle is the magnetic particle described in Patent Document 4, which comprises a base particle containing superparamagnetic particles, a hydrophobic first polymer layer formed on the surface of the base particle, and a second polymer layer formed on the first polymer layer, and the second polymer layer contains polar groups containing one or more atoms of at least one type selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms, introduced by chemically modifying a glycidyl group.

[0031] The mother particles include, for example, particles including a core particle made of a non-magnetic material and a magnetic layer containing superparamagnetic particles provided on the surface of the core particle. The core particle may be made of either an organic or inorganic material, but is preferably made of an organic material. An example of the organic material is a polymer. The polymer is preferably a vinyl polymer, more preferably cross-linked polystyrene or cross-linked polymethyl methacrylate.

[0032] Examples of superparamagnetic particles contained in the magnetic layer include iron oxide particles, such as XFe2O4 (X=Mn, Co, Ni, Mg, Cu, Li) 0.5 Fe 0.5Examples of suitable superparamagnetic particles include ferrite (expressed as FeO, etc.), magnetite (expressed as FeO), or γ-FeO. Among these, preferred examples include superparamagnetic particles containing γ-FeO or FeO, due to their high saturation magnetization and low remanent magnetization. Commercially available superparamagnetic particles (e.g., EXP series manufactured by Ferrotec Corporation) can be used. It is desirable for the superparamagnetic particles to have a hydrophobic surface in terms of affinity and compatibility with the core particles and the monomers that form the polymer layer. Superparamagnetic particles with hydrophobic surfaces can be obtained, for example, by precipitating particles from a fluid containing the superparamagnetic particles with a poor solvent and then washing them. The superparamagnetic particles preferably have a particle diameter of 50 nm or less, more preferably 5 to 30 nm.

[0033] The method for forming a magnetic layer containing the superparamagnetic particles on the surface of the core particles includes, for example, physically adsorbing the superparamagnetic particles to the surface of the core particles. The mass ratio of the core particles to the superparamagnetic particles in the mother particle (core particles:superparamagnetic particles) is preferably 95:5 to 20:80.

[0034] The hydrophobic first polymer layer formed on the surface of the base particles can be formed from a monomer material containing a hydrophobic monomer. The hydrophobic monomer can be either a monofunctional (non-crosslinkable) monomer or a crosslinkable monomer, or a mixture of a monofunctional monomer and a crosslinkable monomer. Examples of the monofunctional monomer include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene; and ethylenically unsaturated carboxylic acid alkyl ester monomers such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, and isobornyl methacrylate. Examples of the crosslinkable monomer include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate; conjugated diolefins such as butadiene and isoprene; divinylbenzene; diallyl phthalate; allyl acrylate; and allyl methacrylate. The proportion of the crosslinkable monomer in the monomers forming the first polymer layer is preferably 1 to 40% by mass, more preferably 5 to 20% by mass. If the proportion of the crosslinkable monomer exceeds 40% by mass, the magnetic particles may become porous, increasing nonspecific adsorption.

[0035] The first polymer layer can be formed on the surface of the base particles by polymerizing a monomer material containing the hydrophobic monomer in a liquid containing, as needed, a polymerization initiator in the presence of the base particles. The thickness of the first polymer layer formed on the base particles is preferably 0.005 to 20 μm, more preferably 0.01 to 5 μm. It is also preferable that the first polymer layer completely covers the base particles.

[0036] The second polymer layer having a glycidyl group is formed primarily for the purpose of introducing functional groups onto the particle surface. The monomer material for forming the second polymer layer includes a glycidyl group-containing monomer. The glycidyl group-containing monomer is preferably a copolymerizable monomer containing a glycidyl group, examples of which include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether. The content of the glycidyl group-containing monomer in the monomer material for the second polymer layer is preferably 20% by mass or more, more preferably 40% by mass or more, and even more preferably 80% by mass or more.

[0037] The monomer material for the second polymer layer preferably further contains a crosslinking monomer. Examples of the crosslinking monomer include those exemplified above for the first polymer layer. The proportion of the crosslinking monomer in the monomer material for the second polymer layer is preferably 1 to 40% by mass, more preferably 5 to 20% by mass. If the proportion of the crosslinking monomer exceeds 40% by mass, the magnetic particles may become porous, which may increase nonspecific adsorption.

[0038] The second polymer layer can be formed by a method essentially similar to that for forming the first polymer layer. That is, the second polymer layer can be formed on the surface of the first polymer layer by polymerizing a monomer material containing the glycidyl group-containing monomer in a liquid to which the aforementioned polymerization initiator and the like are added as needed, in the presence of particles on which the first polymer layer is formed. The thickness of the second polymer layer formed on the first polymer layer can be thinner than that of the first polymer layer, and is preferably 0.005 to 5 μm, more preferably 0.005 to 1 μm.

[0039] The polar group introduced by chemically modifying the glycidyl group of the second polymer layer is preferably a functional group capable of reacting with a ligand, more preferably one containing at least one atom selected from the group consisting of oxygen, nitrogen, and sulfur atoms, and among these, an amino group, an aldehyde group, a carboxy group, or an active ester group is more preferred.

[0040] The glycidyl group may be hydrolyzed along with the introduction of the polar group. Hydrolysis of the glycidyl group generates a 2,3-dihydroxypropyl group. When the second polymer layer contains the 2,3-dihydroxypropyl group in addition to the polar group, nonspecific adsorption of magnetic particles can be reduced.

[0041] Introduction of polar groups into the second polymer layer can be carried out according to the procedure described in Patent Document 4. For example, a method for introducing carboxyl groups includes hydrolyzing glycidyl groups in the second polymer layer and reacting the resulting hydroxyl groups with a carboxylating agent (e.g., a carboxylic acid anhydride or a carboxylic acid chloride). In this case, it is not necessary to esterify all of the hydroxyl groups generated by the hydrolysis of the glycidyl groups; it is preferable that some of the hydroxyl groups remain as hydroxyl groups without being esterified. A carboxylic acid anhydride is preferred as the carboxylating agent. A 1,2-dicarboxylic acid anhydride such as succinic anhydride, maleic anhydride, or phthalic anhydride is preferred as the carboxylic acid anhydride.

[0042] (Immobilization of Ligand) Methods for immobilizing a ligand on the carrier substrate include physical adsorption methods and chemical bonding methods such as covalent bonding and ionic bonding. Physical adsorption methods include a method of directly immobilizing a ligand on a carrier substrate, and a method of chemically bonding the ligand to another protein such as albumin and then adsorbing it to immobilize it on the carrier substrate. Chemical bonding methods include a method of directly bonding the ligand to the carrier substrate using a functional group that is introduced onto the surface of the carrier substrate and is capable of reacting with the ligand, a method of chemically introducing a spacer molecule (such as a carbodiimide compound) between the carrier substrate and the ligand and then bonding them, and a method of bonding a ligand to another protein such as albumin and then chemically bonding the protein to the carrier substrate.

[0043] Therefore, the carrier for separating EVs of the present invention comprises the carrier substrate and the ligand having binding ability to saccharides, immobilized on the carrier substrate. The carrier of the present invention can be used for affinity separation of EVs. Preferably, the carrier of the present invention is a particulate carrier. The particle diameter of the particulate carrier is preferably 1 to 60 μm, more preferably 1 to 10 μm. If the particle size is too small, the yield of EVs may decrease.

[0044] As used herein, the "particle size" of a carrier refers to the volume average particle size measured in accordance with ISO13319:2007, and can be measured using a particle size distribution measuring device conforming to this standard (for example, a Beckman Coulter Multisizer 4e). For example, 1% by mass of particulate carrier dispersed in a 0.1% by mass Tween 20 aqueous solution is added to 100 mL of electrolyte (Beckman Coulter ISOTON II) until the concentration sensor indicates a value of 4 to 10%, and then the particle size of 50,000 particles is measured using a Multisizer 4e, and the average value is calculated, thereby measuring the volume average particle size of the particulate carrier. However, when the particulate carrier is a porous particle, the particle size tends to be underestimated using the above measurement method. Therefore, it is preferable to correct the particle size of porous particles using a shape factor such that the volume average particle size measured by the above measurement method coincides with the value of the volume average particle size measured by a laser diffraction scattering method in accordance with ISO 13320: 2009. Alternatively, when the particulate support is a porous particle, the "particle size" of the support refers to the volume average particle size measured in accordance with ISO 13320: 2009.

[0045] [Method for separating extracellular vesicles (EVs)] In one embodiment, the present invention provides a method for separating EVs. The method for separating EVs of the present invention (hereinafter also referred to as the "method of the present invention") comprises the following steps: contacting a sample containing EVs with the EV separation carrier of the present invention; and contacting the carrier after contact with the sample with a buffer containing a sugar. A schematic diagram showing an overview of the method for separating EVs of the present invention is shown in Figure 1.

[0046] (Sample) EVs separated by the method of the present invention include exosomes, microvesicles, and apoptotic bodies. In a preferred embodiment, EVs separated by the method of the present invention have tetraspanins on their surfaces. Preferably, the tetraspanins include at least one selected from the group consisting of CD9, CD63, and CD81. Preferably, EVs separated by the method of the present invention have a membrane containing tetraspanins, more preferably EVs containing at least one selected from the group consisting of CD9, CD63, and CD81, and even more preferably EVs containing CD63.

[0047] The sample containing EVs used in the method of the present invention is not particularly limited, and examples thereof include various liquids such as body fluids, bacterial cell fluids, cell culture media, cell culture supernatants, and tissue cell lysates. The cells contained in the media, culture supernatants, and lysates may be of eukaryotic or bacterial origin. Among these, body fluids and cell culture supernatants are preferred as the sample. Examples of body fluids include blood components such as whole blood, serum, plasma, blood components, various blood cells, blood clots, platelets, peripheral blood, and umbilical cord blood, as well as sweat, gastric juice, interstitial fluid, joint fluid, synovial fluid, aqueous humor, sputum, pleural effusion, oral mucosa, bone marrow fluid, semen, prostatic fluid, Cowper's gland fluid, pre-ejaculatory fluid, female ejaculate, menstrual fluid, cyst fluid, pericardial fluid, digestive fluid, tissue fluid, body cavity fluid, saliva, bile, vaginal fluid, tears, milk, urine, cerebrospinal fluid, pancreatic juice, nasal fluid, feces, stool, chyme, chyle, ascites, amniotic fluid, pus, sebum, blastocyst cavity fluid, vomit, mucosal secretions, lymph, nasal lavage fluid, and bronchoalveolar lavage fluid, with blood or blood components being preferred. The blood or blood components may be treated with an anticoagulant such as citric acid or EDTA.

[0048] The sample can be collected from any animal that has EVs, such as mammals, humans or non-human primates, dogs, cats, horses, cows, pigs, other farm animals, and rodents (e.g., mice, rats, guinea pigs, etc.). Body fluids collected from these animals or cultures derived from these animals can be used as samples containing EVs, as well as culture supernatants collected from these cultures. Alternatively, the sample can be concentrated or purified in advance by ultrafiltration, ultracentrifugation, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, or the like.

[0049] In a preferred embodiment, the method of the present invention comprises passing the sample containing EVs through an ultrafiltration membrane before contacting the sample with the carrier of the present invention. The molecular weight cutoff (MWCO) of the ultrafiltration membrane is preferably 3 kDa to 800 kDa, more preferably 50 kDa to 800 kDa. Ultrafiltration at such a molecular weight cutoff can achieve high EV separation efficiency. While the reason why ultrafiltration at the above molecular weight cutoff achieves high EV separation efficiency is unclear, it is presumed to be due to the effective elimination of substances that inhibit contact between EVs and the carrier of the present invention. Ultrafiltration of the sample containing EVs can be performed according to conventional procedures for separating or purifying EVs. For example, the sample can be passed through the ultrafiltration membrane, optionally with the addition of a buffer, and the filtrate discarded. This process can be repeated as necessary to separate EVs contained in the sample. Preferably, the EVs in the retentate after the ultrafiltration process are concentrated at least 10-fold compared to before the ultrafiltration process. The ultrafiltration equipment may be of a dead-end type or a cross-flow type.

[0050] (Capturing EVs on a carrier) In the method of the present invention, a sample containing the EVs is contacted with the carrier of the present invention, and the EVs are captured by the ligands of the carrier. The amount of the carrier of the present invention used is preferably 0.00025 to 1 times, and more preferably 0.002 to 0.2 times, the mass of the sample.

[0051] In the method of the present invention, the sample containing EVs is contacted with the carrier of the present invention in a temperature environment preferably ranging from 2 to 42°C, more preferably from 20 to 37°C. The contact time between the sample and the carrier of the present invention is preferably ranging from 10 minutes to 10 hours, more preferably from 10 minutes to 5 hours. The sample is preferably contacted with the carrier in an aqueous medium. Examples of the aqueous medium include water and buffers such as phosphate buffer, HEPES buffer, Tris buffer, carbonate buffer, and MES buffer. The pH conditions during contact between the sample and the carrier of the present invention are not particularly limited, but are preferably in the range of pH 5 to 10, more preferably pH 6 to 8. To maintain such pH conditions, the above-mentioned buffers can be used.

[0052] (Washing of the Carrier) Preferably, the carrier that has come into contact with the sample containing EVs is then washed. This washing can efficiently remove substances unreacted with the carrier and impurities in the sample from the carrier. For example, when the carrier is particulate, washing can be performed by dispersing the carrier particles in a washing solution. On the other hand, when the carrier is in the form of a microplate, the carrier can be washed by contacting the washing solution with the surface of the carrier. More specifically, when the carrier is magnetic particles, the washing step preferably includes a step of collecting the magnetic particles by magnetic force to separate the magnetic particles from the liquid phase, and a step of dispersing the separated magnetic particles in a washing solution. These steps can be repeated as necessary. Examples of the washing solution that can be used include water and buffers such as phosphate buffer, HEPES buffer, Tris buffer, carbonate buffer, and MES buffer.

[0053] (Recovery of EVs) Next, in the method of the present invention, the carrier after contact with the sample as described above is contacted with a buffer solution containing saccharides. Examples of saccharides that can be contained in the buffer solution include at least one saccharide selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, more preferably at least one saccharide selected from the group consisting of oligosaccharides and polysaccharides composed of glucose, glucosamine, or N-acetylglucosamine. Examples of oligosaccharides and polysaccharides that can be contained in the buffer solution include chitobiose, N,N'-diacetylchitobiose, oligoglucosamine (chitosan oligosaccharide), oligo-N-acetylglucosamine (chitin oligosaccharide), etc., preferably N,N'-diacetylchitobiose and oligo-N-acetylglucosamine (chitin oligosaccharide), more preferably oligo-N-acetylglucosamine (chitin oligosaccharide). Examples of the solvent for the saccharide-containing buffer solution include water and buffer solutions such as phosphate buffer, HEPES buffer, Tris buffer, carbonate buffer, and MES buffer. The concentration of the saccharide in the saccharide-containing buffer solution is preferably 1 to 500 mM, more preferably 10 to 200 mM, in terms of monosaccharides. EVs captured on the carrier are dissociated from the ligand by contact with the saccharide-containing buffer solution.

[0054] For example, when the carrier is in the form of particles such as magnetic particles, the carrier particles can be dispersed in the buffer solution. On the other hand, when the carrier is in the form of a microplate, the carrier can be contacted with the buffer solution by adding the buffer solution to the surface of the carrier. More specifically, when the carrier is a magnetic particle, the washed magnetic particles can be collected by magnetic force, and then the collected particles can be dispersed in the buffer solution to dissociate the EVs from the ligand. The magnetic particles in the dispersion can then be collected and removed by magnetic force, allowing the fraction containing EVs to be recovered.

[0055] [Kit for separating extracellular vesicles (EVs)] In one embodiment, the present invention provides a kit for separating EVs. The kit includes the EV separation carrier of the present invention. Preferably, the kit further includes a buffer containing the sugar. Optionally, the kit may further include a washing solution for washing the carrier. Optionally, the kit may further include instructions describing the procedure for the method for separating EVs using the EV separation carrier of the present invention. Optionally, the kit may further include an ultrafiltration membrane for concentrating EVs contained in the sample. Furthermore, when the carrier of the present invention is a magnetic particle, the kit may further include a device for magnetically collecting the magnetic particle, which is the carrier of the present invention.

[0056] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0057] Example 1 (1) Preparation of Magnetic Particles (i) Preparation of Core Particles Two parts by weight of a 75% di(3,5,5-trimethylhexanoyl) peroxide solution (NOF Corporation; "PERLOYL 355-75(S)"; hereinafter referred to as "PERLOYL") was mixed with 20 parts by weight of a 1% by weight aqueous solution of sodium dodecyl sulfate and finely emulsified using an ultrasonic disperser. This mixture was then placed in a reactor containing 13 parts by weight of polystyrene particles with a particle size of 0.77 μm and 41 parts by weight of water, and stirred at 25°C for 12 hours. In a separate vessel, 95 parts by weight of methyl methacrylate (hereinafter referred to as "MMA") and 5 parts by weight of trimethylolpropane trimethacrylate (hereinafter referred to as "TMP") were emulsified in 400 parts by weight of a 0.1% aqueous solution of sodium dodecyl sulfate. The resulting emulsion was then placed in the reactor and stirred at 40°C for 2 hours. The mixture was then heated to 75°C and polymerized for 8 hours. After the reaction solution was cooled to room temperature, the particles were taken out by centrifugation, washed with water, dried and pulverized to obtain core particles A-1.

[0058] (ii) Preparation of Mother Particles Acetone was added to an oil-based magnetic fluid (trade name: "EXP Series," manufactured by Ferrotec Corporation) to precipitate particles, which were then dried to obtain ferrite-based superparamagnetic microparticles (average primary particle diameter: 0.02 μm) having hydrophobized surfaces. Next, the core particles A-1 (15 parts by mass) and 20 parts by mass of the superparamagnetic microparticles were thoroughly mixed in a mixer, and the resulting mixture was treated for 5 minutes using a hybridization system NHS-0 (manufactured by Nara Machinery Works, Ltd.) at a blade (stirring impeller) peripheral speed of 100 m / s (16,200 rpm) to obtain mother particles A-2 having a magnetic layer made of superparamagnetic microparticles on their surfaces.

[0059] (iii) Formation of a polymer layer on the base particles 333 parts by mass of a 0.5% by mass aqueous solution of sodium dodecylbenzenesulfonate was added to a 1 L separable flask, followed by the addition of the base particles A-2 (13.3 parts by mass). The mixture was dispersed using a homogenizer and then heated to 60°C. Separately, a pre-emulsion was prepared by dispersing 18 parts by mass of MMA, 2 parts by mass of TMP, and 0.4 parts by mass of peroyl in 100 parts by mass of a 0.5% by mass aqueous solution of sodium dodecylbenzenesulfonate. This pre-emulsion was added dropwise over 2 hours to the separable flask, the temperature of which had been controlled at 60°C, to form a first polymer layer on the surface of the base particles.

[0060] After the dropwise addition of the pre-emulsion was completed, the separable flask was maintained at 60°C and stirred for 1 hour. Separately, a pre-emulsion was prepared by dispersing 8.75 parts by mass of glycidyl methacrylate, 1.25 parts by mass of TMP, and 0.2 parts by mass of peroyl in 50 parts by mass of a 0.5% by mass aqueous solution of sodium dodecylbenzenesulfonate. This pre-emulsion was added dropwise to the separable flask, the temperature of which was controlled at 60°C, over 1 hour and 20 minutes. The reaction solution was then heated to 75°C, and polymerization was continued for an additional 2 hours to complete the reaction, thereby forming a second polymer layer on the first polymer layer.

[0061] Next, the particles in the separable flask were separated by magnetism and washed with distilled water to obtain magnetic particles A-3 on which a second polymer layer having a glycidyl group was formed.

[0062] (iv) Hydrolysis of glycidyl groups 10 parts by mass of a 1% by mass aqueous solution of sulfuric acid was added to the magnetic particles A-3 (1.0 part by mass), and the particles were dispersed by irradiation with ultrasound for 5 minutes, followed by stirring for 5 hours at 60° C. The magnetic particles were separated from the reaction solution by magnetism and washed five times with pure water to obtain OH group-containing magnetic particles A-4.

[0063] (v) Introduction of Carboxy Groups The magnetic particles A-4 (1.0 part by mass) were washed three times with 1,3-dioxolane and then dispersed in 10 parts by mass of 1,3-dioxolane. A solution of 1 part by mass of succinic anhydride and 0.15 parts by mass of triethylamine was added thereto and stirred at 25°C for 4 hours. After the reaction was completed, the particles were separated by magnetism and washed three times with 1,3-dioxolane and then four times with distilled water to obtain carboxy group-containing magnetic particles (magnetic particles A-5). The obtained magnetic particles A-5 were dispersed in a 0.01% by mass aqueous solution of 2-methyl-4-isothiazolin-3-one adjusted to pH 7.0 using Aldrich ProClin 950 to prepare a dispersion containing 1% by mass of magnetic particles A-5.

[0064] (2) Preparation of Lectin-Immobilized Magnetic Particles 500 μL of a 1% by mass dispersion of magnetic particles A-5 prepared in (1) above was placed in a tube, and the particles were magnetically separated using a magnetic stand. The supernatant was removed from the tube, and the particles were washed three times with 100 mM MES (pH 5). 500 μL of 100 mM MES (pH 5) was added to disperse the particles, to which 0.1 mg of wheat germ lectin (WGA) (Vector Laboratories) and 0.05 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, the particles were magnetically separated, the supernatant was removed, and the particles were washed five times with PBS. The particles were then dispersed in 500 μL of PBS to obtain a dispersion of lectin-immobilized magnetic particles (particle diameter: 3.0 μm).

[0065] (3) Preparation of sample solution containing EVs Five 225 cm samples containing mesenchymal stem cell growth medium (PromoCell) were prepared. 2Human adipose-derived mesenchymal stem cells (PromoCell) were cultured in 100-well culture flasks (Thermo Fischer Scientific) until the cells reached 60-80% cell coverage. The culture supernatant was removed, and the cells were washed three times with PBS. 75 mL of RoosterCollect-EV™ / EV Pro™ (RoosterBio) was added per flask and incubated at 37°C and 5% CO2 for 48 hours. The culture supernatant was then collected. The collected culture supernatant was centrifuged at 300 x g for 10 minutes, and the supernatant was further centrifuged at 12,000 x g for 30 minutes. The supernatant was collected and filtered through a 0.22 μm filter (Merck), followed by concentration by ultrafiltration (Merck Amicon® Ultra-15, MWCO = 100 kDa) and the addition of PBS to the pre-concentration volume. This procedure was repeated 10 times. Finally, a sample solution containing EVs 20-fold concentrated relative to the pre-concentration volume was obtained.

[0066] (4) Separation of EVs using lectin-immobilized magnetic particles The lectin-immobilized magnetic particle dispersion prepared in (2) above was diluted to a particle concentration of 0.2% (w / v). 1 mL of this diluted solution was placed in a 2 mL tube, and the particles were magnetically separated to remove the solvent. 1 mL of the sample solution containing EVs prepared in (3) above was added to this, and the mixture was shaken at 25°C for 1 hour to allow EVs to bind to the lectin-immobilized particles. The particles were magnetically separated, and the remaining liquid was collected as the unadsorbed fraction. Next, 1 mL of PBS was added, and the mixture was shaken at 25°C for 1 hour to wash the particles. The particles were then magnetically separated, and the remaining liquid was collected as the washed fraction. Next, 1 mL of PBS containing oligo-N-acetylglucosamine (Tokyo Chemical Industry Co., Ltd.) was added to the particles as an eluent, and the mixture was shaken at 25°C for 1 hour to dissociate EVs from the particles. Thereafter, the particles were magnetically separated, and the remaining liquid was collected as an eluted fraction to obtain an EVs solution.

[0067] Example 2 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the polystyrene particles used in (1)(i) of Example 1 were changed to particles with a particle diameter of 0.40 μm, and the particle diameter of the lectin-immobilized magnetic particles obtained in (2) was 1.6 μm. An elution fraction (EVs solution) was obtained using these magnetic particles.

[0068] Example 3 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the polystyrene particles used in (1)(i) of Example 1 were changed to particles with a particle diameter of 2.9 μm, and the particle diameter of the lectin-immobilized magnetic particles obtained in (2) was 10 μm. An elution fraction (EVs solution) was obtained using these particles.

[0069] Example 4 (1) Preparation of Porous Particles 3.58 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 360 g of pure water, and the mixture was heated and stirred to dissolve the polyvinyl alcohol. After cooling, 0.36 g of sodium dodecyl sulfate (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.36 g of sodium sulfate (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), and 0.18 g of sodium nitrite (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added and stirred to obtain aqueous solution A. A monomer composition consisting of 12.00 g of glycidyl methacrylate (manufactured by Mitsubishi Chemical Corporation) and 1.33 g of divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.) was dissolved in 24.43 g of diisobutyl ketone (manufactured by Mitsui Chemicals, Inc.) to prepare monomer solution B.

[0070] The entire amount of the aqueous solution A was placed in a separable flask, which was then fitted with a thermometer, stirring blades, and a condenser. The flask was then placed in a hot water bath and stirred under a nitrogen atmosphere. The entire amount of the monomer solution B was placed in the separable flask and heated in the hot water bath. When the internal temperature reached 85°C, 0.53 g of 2,2'-azobisisobutyronitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the reaction solution was then stirred for 3 hours while maintaining the temperature at 86°C. The reaction solution was then cooled, filtered, and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. The particles were then dispersed in pure water to a particle concentration of 10% by mass, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1." The particle diameter of porous particles 1 was 50 μm.

[0071] 16 mL of the dispersion of porous particles 1 was added to an aqueous solution (pH 8.3) containing 0.1 M sodium sulfate and 0.5 M sodium thioglycolate, and the mixture was shaken and stirred at 25° C. for 5 hours. The reaction solution was filtered, the filtrate was removed, and the mixture was washed four times with pure water. The particles were then dispersed in pure water to a particle concentration of 1% by mass, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 2."

[0072] (2) Preparation of Lectin-Immobilized Porous Particles: 500 μL of the 1% by weight aqueous dispersion of porous particles 2 obtained in (1) above was placed in a 0.45 μm filter tube, centrifuged to remove the flow-through, and the particles were washed three times with a 100 mM MES aqueous solution (pH 5). 500 μL of 100 mM MES (pH 5) was added to disperse the particles, to which 0.1 mg of wheat germ lectin (WGA) (Vector Laboratories) and 0.05 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fujifilm Wako Pure Chemical Industries, Ltd.) were added and stirred at room temperature for 5 hours. After the reaction was completed, the flow-through was removed by centrifugation, and the particles were washed five times with PBS buffer and then dispersed in 500 μL of PBS to obtain a dispersion of lectin-immobilized porous particles.

[0073] (3) Separation of EVs Using Lectin-Immobilized Porous Particles The lectin-immobilized porous particle dispersion prepared in (2) above was diluted to a particle concentration of 0.2% (w / v). 1 mL of this diluted solution was placed in a 0.45 μm filter tube and centrifuged to remove the permeated solvent. 1 mL of the sample solution containing EVs prepared in (3) of Example 1 was added to this, and the mixture was shaken at 25°C for 1 hour to allow EVs to bind to the lectin-immobilized particles. The filter tube containing the reaction solution was centrifuged, and the flow-through was collected as the unadsorbed fraction. 1 mL of PBS buffer was added to the tube, and the mixture was shaken at 25°C for 1 hour to wash the particles, followed by centrifugation to collect the flow-through as the wash fraction. Next, 1 mL of PBS containing oligo-N-acetylglucosamine was added to the tube, and the mixture was shaken at 25°C for 1 hour to dissociate EVs from the particles. The tube was then centrifuged, and the flow-through was collected as the elution fraction to obtain an EVs solution.

[0074] Example 5 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the ultrafiltration membrane used to concentrate the sample solution containing EVs in (3) of Example 1 was changed to one with a MWCO of 3.5 kDa, and an eluted fraction (EVs solution) was obtained using these particles.

[0075] Example 6 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the lectin immobilized on the magnetic particles in (2) of Example 1 was changed to tomato-derived lectin (LEL) (Vector Laboratories), and an elution fraction (EVs solution) was obtained using these magnetic particles.

[0076] Example 7 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the lectin immobilized on the magnetic particles in (2) of Example 1 was changed to potato-derived lectin (STL) (Vector Laboratories), and an elution fraction (EVs solution) was obtained using these magnetic particles.

[0077] Example 8 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that the lectin immobilized on the magnetic particles in (2) of Example 1 was changed to Datura stramonium lectin (DSL) (Vector Laboratories), and an elution fraction (EVs solution) was obtained using these particles.

[0078] Example 9 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that in (4) of Example 1, PBS in which N-acetylglucosamine (Tokyo Chemical Industry Co., Ltd.) was dissolved was used as the eluent, and an eluted fraction (EVs solution) was obtained using these particles.

[0079] Example 10 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1 (4), except that PBS containing N,N'-diacetylchitobiose (Tokyo Chemical Industry Co., Ltd.) was used as the eluent, and an eluted fraction (EVs solution) was obtained using these particles.

[0080] Example 11 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1 (4), except that PBS containing dissolved oligoglucosamine (Tokyo Chemical Industry Co., Ltd.) was used as the eluent, and an eluted fraction (EVs solution) was obtained using these particles.

[0081] Comparative Example 1 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that ultrafiltration of the supernatant was not performed in (3) of Example 1, and PBS was used as the eluent in (4), and an eluted fraction was obtained using these.

[0082] Comparative Example 2 Lectin-immobilized magnetic particles were prepared in the same manner as in Example 1, except that in (4) of Example 1, PBS in which mannose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved was used as the eluent, and an eluted fraction was obtained using these.

[0083] Test Example 1 Evaluation of EVs Yield (1) Evaluation of Particle Count The number of particles contained in the sample solution containing EVs obtained in Example 1(3), as well as in the unadsorbed fraction, washed fraction, and eluted fraction obtained in each Example and Comparative Example, was counted using a nanoparticle analyzer (NanoSight LM10, manufactured by NanoSight).

[0084] (2) Quantification of Tetraspanins by ELISA The co-expression levels of CD9 and CD63, which are tetraspanins found to be expressed in EVs, were quantified using a CD9 / CD63 ELISA kit (manufactured by Cosmo Bio Co., Ltd.) for the sample solution containing EVs obtained in (3) of Example 1, and the unadsorbed fraction, washed fraction, and eluted fraction obtained in each Example and Comparative Example. The obtained CD9 / CD63 quantification value was divided by the number of particles counted in (1) above to determine the CD9 / CD63 co-expression level per particle.

[0085] The results are shown in Tables 1 and 2. In Examples 1 to 11, the ratio of particle number in the elution fraction to the sample solution (particle yield) was approximately 10% to 22%. On the other hand, in Examples 1 to 11, the amount of CD9 / CD63 per particle in the elution fraction was approximately three times that of the sample solution, indicating that EVs were selectively separated in the elution fraction. In contrast, in Comparative Examples 1 and 2, the particle yield was approximately 1% or less, and the amount of CD9 / CD63 per particle was below the lower limit of quantitation (3.1 pg / mL) of the ELISA kit, indicating that almost no EVs were separated. Furthermore, in Example 5, which used an ultrafiltration membrane with a smaller pore size than Example 1, and Comparative Example 1, in which ultrafiltration was not performed, the residual rate of CD9 / CD63 in the unadsorbed fraction relative to the sample solution was high, suggesting that sample concentration or purification by ultrafiltration under appropriate conditions may improve EVs binding to lectin-immobilized particles.

[0086] [Test Example 2] Evaluation of the yield of tetraspanin-containing EVs by CLEIA The tetraspanins CD9, CD63, and CD81 expressed in EVs were quantified by chemiluminescent enzyme immunoassay (CLEIA) for the sample solution containing EVs obtained in (3) of Example 1, and the unadsorbed fraction, washed fraction, and eluted fraction obtained in (4). Specifically, streptavidin-labeled magnetic particles (MS300 / Streptavidin, manufactured by JSR Life Sciences Corporation) were conjugated to anti-CD9 biotinylated antibody (manufactured by MBL), anti-CD63 biotinylated antibody (manufactured by MBL), or anti-CD81 biotinylated antibody (manufactured by MBL) to obtain antibody-bound magnetic particles. TBS (pH 7.4) containing 0.2% (w / v) of the antibody-bound magnetic particles and 0.1% (w / v) of a nonionic surfactant (Pluronic F-68) was added in an amount of 25 μL to each well of a 96-well white plate (manufactured by Corning Incorporated) and mixed. 25 μL of the sample solution, unadsorbed fraction, washed fraction, or eluted fraction was added to this well, mixed, and shaken at 25°C for 20 minutes. After the reaction, the antibody-bound magnetic particles were washed with a washing solution (TBS containing 0.01% (w / v) Tween 20). After removing the washing solution, 50 μL of a solution containing alkaline phosphatase-labeled antibody was added to each well, and the plate was shaken at 25°C for 20 minutes. The alkaline phosphatase-labeled antibodies used were alkaline phosphatase-labeled anti-CD9 antibody (manufactured by MBL) for the anti-CD9 antibody magnetic particles, alkaline phosphatase-labeled anti-CD63 antibody (manufactured by MBL) for the anti-CD63 antibody magnetic particles, and alkaline phosphatase-labeled anti-CD81 antibody (manufactured by MBL) for the anti-CD81 antibody magnetic particles. The antibody solution provided with the product was diluted 2000-fold with TBS containing 0.01% (w / v) Tween 20. After the reaction, the antibody-bound magnetic particles were washed with a washing solution (TBS containing 0.01% (w / v) Tween 20), and then 50 μL of luminescent substrate solution (manufactured by LSI Medience) was added. After 5 minutes, the luminescence intensity was measured using a luminometer (CYTATION 5, manufactured by BioTek).

[0087] The results are shown in Table 3. Tetraspanins CD9, CD63, and CD81 were all detected in the eluted fractions, and their luminescence intensities (concentrations) were all 70% or more of those in the sample solution, indicating that EVs containing these tetraspanins were recovered in high yields in the eluted fractions.

[0088]

[0089]

[0090]

[0091] [Test Example 3] Evaluation of the physiological activity of the obtained EVs on cells The EVs obtained using the lectin-immobilized magnetic particles prepared in Example 1 were evaluated for their TNF-α suppression effect in macrophage-like cells.

[0092] (1) Preparation of sample solution containing EVs Twenty-five 150 cm EVs containing mesenchymal stem cell growth medium (PromoCell) were prepared. 2 Human adipose-derived mesenchymal stem cells (PromoCell) were cultured in 100ml culture dishes (Sumitomo Bakelite Co., Ltd.) until the cells reached 60-80% of the culture area. The culture supernatant was removed, and the cells were washed three times with PBS. 20 mL of RoosterCollect-EV™ / EV Pro™ (RoosterBio) was added per dish, and the cells were incubated at 37°C and 5% CO2 for 48 hours, after which the culture supernatant was recovered. The collected culture supernatant was pooled and centrifuged at 300 x g for 10 minutes. The supernatant was then further centrifuged at 12,000 x g for 30 minutes. The supernatant was collected and filtered through a 0.22 μm filter (Merck), then concentrated by ultrafiltration (Hansa BioMed TFF-EVs small, MWCO = 800 kDa), and then washed with 1000 mL of PBS (Fujifilm Wako Pure Chemical Industries, Ltd.). Finally, a sample solution containing EVs 50-fold concentrated was obtained.

[0093] (2) Preparation of EVs Solution An EVs solution (elution fraction) was obtained from the sample solution containing EVs prepared in (1) by the procedure of (4) of Example 1, and designated as Solution A. From Solution A, an ultrafiltration membrane (Amicon (registered trademark) Ultra-0.5, Ultracel-100, MWCO = 100 kDa, manufactured by Merck) was used to separate only components of 100 kDa or less, and a fraction (filtrate) was obtained and designated as Solution B. The eluate used in (4) of Example 1 (PBS containing dissolved oligo-N-acetylglucosamine) was designated as Solution C. From the sample solution prepared in (1), an EVs solution was obtained by a separation method using magnetic particles equipped with phosphatidylserine-binding protein as a ligand, using MagCapture (trademark) Exosome Isolation Kit PS Ver. 2 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and designated as Solution D. An ultrafiltration membrane (Merck Amicon® Ultra-0.5, Ultracel-100, MWCO = 100 kDa) was used to separate only components of 100 kDa or less from Solution D, obtaining a fraction (filtrate) designated Solution E. The eluate used in the separation using the MagCapture™ Exosome Isolation Kit PS Ver. 2 was designated Solution F. In a test to examine the physiological activity of the obtained EVs on macrophage-like cells, the amount of EVs in each EV solution was standardized to perform a comparative test using the same amount of EVs. The amount of EVs in each of Solutions A and D was measured using ELISA against CD9 / CD63 (Cosmo Bio Co., Ltd.), and the amount of EVs in each solution was adjusted to 165 pg / mL.

[0094] Dexamethasone is used clinically as a standard therapeutic agent to suppress inflammation and is known to suppress the expression of TNF-α (Oncotarget, 2017, Vol. (30), pp:49735-49748, doi:10.18632 / oncotarget.17683). Dexamethasone (20 μg / mL) was dissolved in PBS containing 4% DMSO (Solution G) and PBS containing 4% DMSO (Solution H). Additionally, solutions containing only PBS, Solution I, and Solution J were prepared.

[0095] (3) Administration of EV solution to macrophage-like cells RAW264.7 cells were suspended in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Gibco), and 2 × 10 5 500 μL of cells were seeded into a 24-well plate so that there were 100 cells / well. After 2 hours of culture, 25 μL of medium was removed from each well, and 25 μL of any of solutions A to J prepared in (2) above was added to each well. After 24 hours of culture, 10 μg / mL lipopolysaccharide (LPS) (MilliporeSigma) dissolved in PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the wells containing solutions A to I to a final concentration of 100 ng / mL. It is known that the addition of LPS to macrophage-like cells increases TNF-α expression (Stem Cell Research & Therapy, 2021, 12:519, doi:10.1186 / s13287-021-02591-4). Wells containing Solution J were treated with LPS-free PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) as a control (final LPS concentration: 0 ng / mL) that did not induce TNF-α expression. After 24 hours of culture, cells were harvested and mRNA was extracted using the RNeasy Plus Mini Kit (Qiagen). After reverse transcription using SuperScript® VILO™ Master mix, quantitative PCR (Thermo Fisher Scientific, 7500 Fast Realtime PCR System) was performed using TaqMan™ Fast Advanced Master mix (Thermo Fisher Scientific). Quantitative PCR was performed using TaqMan Probes (Thermo Fisher Scientific) for the housekeeping gene GAPDH and the target gene TNF-α. The quantitative value of TNF-α mRNA was calculated using the ΔΔCt method. The relative expression levels of TNF-α mRNA in macrophage-like cells after addition of each solution are shown in Figure 2.

[0096] Addition of LPS to macrophage-like cells increased the TNF-α mRNA expression level from 1.00 (solution J: final LPS concentration 0 ng / mL) to 6.25 (solution I: final LPS concentration 100 ng / mL) in relative terms. The TNF-α mRNA expression level increased by the addition of LPS was reduced by the addition of dexamethasone (solution G). In cells treated with solution A, the TNF-α mRNA expression level was equivalent to the mRNA expression level in cells without LPS (solution J: relative amount 1.00), in which TNF-α was not induced. This indicates that the addition of solution A reduced the TNF-α mRNA expression level induced by the addition of LPS to the TNF-α mRNA expression level in cells without LPS. Furthermore, the inhibitory effect of Solution A on TNF-α mRNA expression was more pronounced than that achieved by the addition of dexamethasone (Solution G). Addition of Solutions B and C did not result in a decrease in TNF-α mRNA expression (Solution B: relative amount 6.94, Solution C: relative amount 6.27). This suggests that the inhibitory effect of Solution A on TNF-α mRNA expression is due to EVs contained in Solution A, rather than to small molecule or protein contamination or eluate components. In cells treated with Solution D, the level of TNF-α mRNA expression was comparable to that of cells treated with LPS (Solution D: relative amount 5.96, Solution I: 6.25), indicating that Solution D did not have an inhibitory effect on TNF-α mRNA expression.

[0097] This test example demonstrated that EVs isolated using an eluate containing lectin-immobilized magnetic particles and sugars exhibit an inhibitory effect on TNF-α expression in macrophage-like cells, and are expected to have a strong anti-inflammatory effect. The results of Test Example 3 demonstrate that EVs isolated by the method of the present invention are physiologically active in living organisms and function in vivo. The effect of the method of the present invention, in which isolated EVs are physiologically active and function in vivo, is an extremely significant effect not demonstrated by prior art.

Claims

1. A method for separating extracellular vesicles, comprising: contacting a sample containing extracellular vesicles with a carrier having a ligand for capturing extracellular vesicles; and contacting the carrier, after contact with the sample, with a buffer solution containing sugars, wherein the sugars are at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, and the ligand has a binding ability to the sugars.

2. The method according to claim 1, wherein the saccharide is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine.

3. The method of claim 1, wherein the ligand is a lectin.

4. The method according to claim 3, wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin.

5. The method of claim 1, further comprising washing the carrier after contact with the sample before contacting it with the buffer solution containing the sugar.

6. The method of claim 1, further comprising passing said sample through an ultrafiltration membrane before contacting said sample with said carrier.

7. The method of claim 1, wherein the extracellular vesicles have tetraspanins on their surfaces.

8. The method of claim 7, wherein the tetraspanins include at least one selected from the group consisting of CD9, CD63, and CD81.

9. The method according to claim 1, wherein the carrier is a particulate carrier having a particle size of 1 to 60 μm.

10. A carrier for separating extracellular vesicles, comprising: a carrier substrate; and a ligand immobilized on the carrier substrate, wherein the carrier is brought into contact with a sample containing extracellular vesicles, and then brought into contact with a buffer containing sugars, thereby dissociating the extracellular vesicles captured on the carrier from the ligand, thereby separating the extracellular vesicles, wherein the sugar is at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom, and the ligand has a binding ability to the sugar.

11. The carrier according to claim 10, wherein the saccharide is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine.

12. The carrier according to claim 10, wherein the ligand is a lectin.

13. The carrier according to claim 12, wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin.

14. The carrier according to claim 10, wherein the extracellular vesicles have tetraspanin on their surface.

15. The carrier according to claim 14, wherein the tetraspanin comprises at least one selected from the group consisting of CD9, CD63, and CD81.

16. The carrier according to claim 10, which is a particulate carrier having a particle size of 1 to 60 μm.

17. A kit for separating extracellular vesicles, comprising: an extracellular vesicle separation carrier; and a buffer solution containing sugars; the extracellular vesicle separation carrier comprising: a carrier substrate; and a ligand immobilized on the carrier substrate, the ligand having a binding ability to the sugars; the buffer solution containing the sugars is used to dissociate the extracellular vesicles captured on the extracellular vesicle separation carrier from the ligand; and the sugar is at least one selected from the group consisting of glucose, glucosamine, N-acetylglucosamine, and oligosaccharides and polysaccharides derived therefrom.

18. The kit according to claim 17, wherein the saccharide is at least one selected from the group consisting of N-acetylglucosamine, chitobiose, N,N'-diacetylchitobiose, oligoglucosamine, and oligo-N-acetylglucosamine.

19. The kit of claim 17, wherein the ligand is a lectin.

20. The kit according to claim 19, wherein the lectin comprises at least one selected from the group consisting of wheat germ-derived lectin, tomato-derived lectin, potato-derived lectin, and Datura stramonium-derived lectin.

21. The kit of claim 17, wherein the extracellular vesicles have tetraspanins on their surfaces.

22. The kit of claim 21, wherein the tetraspanin comprises at least one selected from the group consisting of CD9, CD63, and CD81.

23. The kit according to claim 17, wherein the carrier for separating extracellular vesicles is a particulate carrier having a particle diameter of 1 to 60 μm.

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