Method for collecting extracellular vesicles

Abstract

The present invention provides a method that makes it possible to efficiently harvest extracellular vesicles, the method being an alternative to conventional techniques. More specifically, the present invention provides an extracellular vesicle harvesting method that includes the following steps (a) and (b). Step (a): Mixing (i) an extracellular vesicle-containing specimen, (ii) particles with a substance immobilized thereon, the substance having affinity with extracellular vesicles, and (iii) a polymer, to obtain a mixed solution that includes (i') target particles bonded to extracellular vesicles via said substance and (ii') the polymer. Step (b): Isolating the target particles from the mixed solution.　It is preferable that a step for lowering the viscosity of the mixed solution is further included between the steps (a) and (b).

Description

[Technical Field]

[0001] The present invention relates to a method for recovering extracellular vesicles. [Background technology]

[0002] Extracellular vesicles (EVs) are tiny membrane-structured vesicles secreted by various cells and found in body fluids such as blood or cell culture media. Extracellular vesicles secreted outside cells include exosomes, ectosomes, and apoptotic vesicles. Because EVs are a diverse group containing various substances that play a role in intercellular signaling, they are analyzed for diagnostic and drug discovery purposes. Therefore, there is a need for the development of methods for recovering EVs useful for such analyses. For example, Patent Document 1 describes a method for recovering EVs using a chelating agent. Patent Document 2 describes a method for isolating EVs by centrifuging an EV-containing sample in the presence of two polymers, including polyvinylpyrrolidone, to form a bilayer between the two polymers, and concentrating the EVs at the interface between the two layers. Patent Document 3 describes a method for isolating extracellular vesicles by co-precipitation of extracellular vesicles using an extracellular matrix-forming polymer containing polyvinylpyrrolidone, in which a polycationic substance is co-present. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2018 / 070479 [Patent Document 2] US Patent Application Publication No. 2018 / 0164197 [Patent Document 3] International Publication No. 2017 / 178472 Summary of the Invention [Problem to be solved by the invention]

[0004] If extracellular vesicles could be recovered from samples containing extracellular vesicles with high efficiency, it would be promising for applications in diagnosis, drug discovery, etc. However, conventional methods have the problem that they cannot always recover extracellular vesicles with high efficiency.

[0005] Therefore, the object of the present invention is to develop an alternative method to the prior art that allows for highly efficient recovery of extracellular vesicles. [Means for solving the problem]

[0006] After extensive research, the present inventors have found that a method using a polymer and particles (solid phase) in combination can recover extracellular vesicles with high efficiency. More specifically, (a) (i) a sample containing extracellular vesicles, (ii) particles to which a substance having affinity for the extracellular vesicle membrane is immobilized, and (iii) a polymer are mixed to obtain a mixture containing (i') target particles bound to the extracellular vesicles via the substance, and (ii') a polymer; and (b) separating the target particles from the mixture, thereby recovering extracellular vesicles with high efficiency. The above-mentioned prior art neither describes nor suggests a method using a polymer and particles (solid phase) in combination.

[0007] The present inventors have also found that extracellular vesicles can be recovered more efficiently by reducing the viscosity of the mixture before separating particles from the mixture. Ultrasonic treatment has traditionally been used to disrupt lipid bilayer membranes (e.g., cell membranes) in operations such as homogenization. Therefore, it was expected that ultrasonic treatment to reduce the viscosity of a mixture containing extracellular vesicles with lipid bilayer membranes would cause the destruction of the extracellular vesicles, resulting in a decrease in the recovery rate of the extracellular vesicles. However, the present inventors have unexpectedly found that even when ultrasonic treatment is performed to reduce the viscosity of a mixture containing extracellular vesicles, the recovery rate of the extracellular vesicles can be improved, and have completed the present invention.

[0008] That is, the present invention is as follows. [1] A method for recovering extracellular vesicles, comprising the following (a) and (b): (a) mixing (i) an extracellular vesicle-containing sample, (ii) particles to which a substance having affinity for the extracellular vesicle membrane is immobilized, and (iii) a polymer to obtain a mixture containing (i') target particles bound to the extracellular vesicles via the substance, and (ii') the polymer; and (b) separating the target particles from the mixture; [2] The method of [1], including the following (a) to (c): (a) mixing (i) a sample containing extracellular vesicles, (ii) particles having immobilized thereon a substance having affinity for extracellular vesicle membranes, and (iii) a polymer to obtain a mixture containing (i') target particles bound to extracellular vesicles via the substance, and (ii') a polymer; (b) reducing the viscosity of the mixture; and (c) separating the target particles from the solution obtained in (b). [3] The method of [1] or [2], comprising, after separation of the target particles, (I) washing the target particles and / or (II) releasing extracellular vesicles from the target particles. [4] The method according to [2] or [3], wherein the viscosity is reduced by treatment with ultrasound or an enzyme having polymer-decomposing ability. [5] The method of [4], wherein the ultrasonic wave is a dry type or a water bath type. [6] Method [4] or [5], wherein the ultrasonic frequency is in the range of 40 kHz or less or 950 kHz or more. [7] The method according to any one of [1] to [6], wherein the polymer is a polysaccharide, a protein, or a polyvinyl derivative having a carbonyl-containing hydrophilic group. [8] The method according to [7], wherein the polysaccharide is a cellulose derivative in which the hydrogen atom of at least one hydroxyl group in cellulose is substituted with a carboxyalkyl or hydroxyalkyl. [9] The method according to any one of [1] to [8], wherein the polymer has a weight-average molecular weight of 10 kDa or more.

[10] The method according to any one of [1] to [9], wherein the polymer concentration in the mixed solution (a) is 0.01 to 10.00% by weight.

[11] Any of the methods [1] to

[10] , further comprising mixing (iv) a chelating agent in (a).

[12] Any of the methods described in [1] to

[11] , wherein the substance having affinity for the extracellular vesicle membrane is an antibody against a tetraspanin membrane protein or an antibody against an extracellular matrix metalloproteinase inducer.

[13] Any of the methods [1] to

[12] , wherein the sample containing extracellular vesicles is an animal-derived liquid sample or a culture supernatant sample.

[14] A method for analyzing extracellular vesicles, comprising the following (1) and (2): (1) Separating extracellular vesicles from an extracellular vesicle-containing sample by any of the methods [1] to

[13] ; and (2) To analyze isolated extracellular vesicles.

[15] A method for producing a polymer-degrading composition comprising: (a) a polymer; (b) a substance having an affinity for an extracellular vesicle membrane; and (c) an enzyme having polymer-degrading ability; the substance is in free form or immobilized on a particle, If the substance is in free form, the kit may further comprise particles.

[16] The kit according to

[15] , wherein the polymer is a polysaccharide or a protein, and the enzyme capable of decomposing the polymer is a glycolytic enzyme or a protease. [Effects of the Invention]

[0009] According to the present invention, extracellular vesicles can be recovered with higher efficiency. Therefore, compared to conventional methods, the present invention makes it possible to rapidly prepare a desired amount of extracellular vesicles and efficiently prepare large amounts of extracellular vesicles. Furthermore, according to the present invention, extracellular vesicles can be recovered with high purity. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD9 antibody of serum specimens diluted with PBS, EDTA / EGTA-PBS ("ED / EG"), or various concentrations of CMC-PBS. [Figure 2] Figure 2 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD9 antibody from serum samples diluted with PBS or various concentrations of CMC-PBS at 4°C overnight or at 37°C for 1 hour. [Figure 3] Figure 3 shows the particle count results measured by nanoparticle tracking analysis for samples obtained by immunoprecipitation with anti-CD9 antibody from serum specimens diluted with PBS, EDTA / EGTA-PBS ("ED / EG"), or CMC-PBS. [Figure 4] Figure 4 shows Western blotting using biotinylated anti-CD9 antibodies of samples obtained by immunoprecipitation with anti-CD9 antibodies from serum and plasma containing five types of anticoagulants (heparin, EDTA, citrate, ACD (acid-citrate-dextrose), and CPD (citrate phosphate-dextrose)) diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 5A] Figure 5A shows Western blotting using anti-tetraspanin membrane protein antibodies (anti-CD63 antibody and anti-CD81 antibody) of samples obtained by immunoprecipitation with anti-tetraspanin membrane protein antibodies (anti-CD63 antibody and anti-CD81 antibody) of serum specimens diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 5B]Figure 5B shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD147 antibody from serum specimens diluted in PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 6] Figure 6 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD9 antibody from body fluids (urine and saliva, two samples each, designated "#1" and "#2") diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 7A] FIG. 7A shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HEC-PBS, or CMC-PBS. [Figure 7B] FIG. 7B shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HPC-PBS, or CMC-PBS. [Figure 7C] Figure 7C shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HPMC-PBS, or CMC-PBS. [Figure 8] FIG. 8 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS or various concentrations of PVP-PBS. [Figure 9A] Figure 9A shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation of serum specimens diluted with PBS or CMC-PBS with anti-CD9 antibody at temperatures ranging from 35 to 60°C. [Figure 9B]FIG. 9B shows Western blotting using anti-CD63 antibody of samples obtained by immunoprecipitation of serum specimens diluted with CMC-PBS with anti-CD63 antibody at temperatures ranging from 35°C to 60°C. [Figure 9C] FIG. 9C shows Western blotting using anti-CD81 antibody of samples obtained by immunoprecipitation of serum specimens diluted with CMC-PBS with anti-CD81 antibody at temperatures ranging from 35°C to 60°C. [Figure 10] Figure 10 shows the amount of EML4-ALK mRNA detected in samples obtained by immunoprecipitation with anti-CD9 antibody or anti-CD63 antibody from culture supernatants of human lung cancer cells H2228 diluted with PBS, CMC-PBS ("CMC"), EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C") (expressed as fold change relative to samples diluted with PBS). [Figure 11] Figure 11 shows a relative comparison of the amount of residual magnetic particles when carboxymethyl cellulose (CMC) was used. Cellulase: Cellulase treatment; Non-US: No ultrasonic treatment. The ultrasonic frequencies used were 100 kHz, 200 kHz, 950 kHz, and 1.6 MHz. [Figure 12A] 12A is a diagram showing a relative comparison of the amount of residual magnetic particles when CMC is used. Non-US: no ultrasonic treatment; US: ultrasonic treatment (30 kHz). [Figure 12B] Figure 12B shows a relative comparison of CD9 counts when CMC was used. Non-US: no ultrasonic treatment; US: ultrasonic treatment (30 kHz). [Figure 12C] Figure 12C shows a comparison of EV recovery efficiencies based on CD9 detection by Western blotting using anti-CD9 antibodies for magnetic particles recovered from CMC-containing solutions by magnetic collection. Non-US: no sonication; US: sonication (30 kHz). [Figure 13]Figure 13 shows a relative comparison of the amount of residual magnetic particles when CMC was used. Cellulase: cellulase treatment; Non-US: no ultrasonic treatment. Solutions containing CMC at different final concentrations were used as the CMC-containing solutions. [Figure 14] Figure 14 shows a relative comparison of the amount of residual magnetic particles when cellulose derivatives other than CMC were used. Cellulase: cellulase treatment; Non-US: no ultrasonic treatment. Hydroxypropyl cellulose 80 kDa (HPC80K) or hydroxyethyl cellulose 380 kDa (HEC380K) (Drich) were used as cellulose derivatives at different final concentrations. [Figure 15] Figure 15 shows a relative comparison of the amount of residual magnetic particles after different ultrasonic treatments. Cellulase: Cellulase treatment; Non-US: No ultrasonic treatment. The ultrasonic treatment was performed at a frequency of 30 kHz (output power of 10W, 20W, or 35W) or 40 kHz (output power of 70W). [Figure 16] FIG. 16 shows a relative comparison of EV recovery efficiency based on CD9 detection by Western blotting using an anti-CD9 antibody for magnetic particles recovered by magnetic collection after glycolytic enzyme treatment. [Figure 17] 17 shows nanoparticle tracking analysis of extracellular vesicles released from magnetic particles. Non-US: no sonication; CMC: sonication (with CMC); HEC: sonication (with HEC). [Figure 18] Figure 18 shows a relative comparison of EV recovery efficiency based on CD9 detection by Western blotting using anti-CD9 antibodies for magnetic particles recovered from a cellulose derivative-containing solution by magnetic collection. Non-US: no ultrasonic treatment; CMC: carboxymethylcellulose; HEC: hydroxyethylcellulose. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Extracellular vesicle collection method The present invention provides a method for recovering extracellular vesicles.

[0012] Extracellular vesicles are minute vesicles with a membrane structure secreted by various cells. Examples of extracellular vesicles include exosomes, ectosomes, and apoptotic vesicles. Preferably, the extracellular vesicles are exosomes. Extracellular vesicles can also be defined by their size. The size of extracellular vesicles is, for example, 30 to 1000 nm, preferably 50 to 300 nm, and more preferably 80 to 200 nm. The size of extracellular vesicles can be measured by, for example, a method based on the Brownian motion of extracellular vesicles, a light scattering method, an electrical resistance method, or the like. Preferably, the size of extracellular vesicles is measured using a NanoSight (Malvern Instruments).

[0013] The recovery method of the present invention comprises the following steps (a) and (b): (a) (i) a sample containing extracellular vesicles, (ii) particles to which a substance having affinity for extracellular vesicle membranes is immobilized, and (iii) a polymer are mixed to obtain a mixture containing (i') target particles bound to the extracellular vesicles via the substance, and (ii') the polymer; and (b) separating the target particles from the mixture;

[0014] In the above step (a), (i) a sample containing extracellular vesicles, (ii) particles to which a substance having affinity for the extracellular vesicle membrane is immobilized, and (iii) a polymer are mixed. By mixing, the extracellular vesicles in the sample (i) bind to the substance having affinity for the extracellular vesicle membrane immobilized on the particles (ii), generating target particles bound to the extracellular vesicles via the substance. Thus, a mixture containing (i') target particles bound to the extracellular vesicles via the substance having affinity for the extracellular vesicle membrane and (ii') a polymer is obtained.

[0015] In the above step (a), the mixing of (i) to (iii) is not particularly limited as long as a mixture containing (i') target particles bound to extracellular vesicles via the substance and (ii') a polymer is obtained. For example, (i) to (iii) can be mixed simultaneously or separately. When (i) to (iii) are mixed separately, two of (i) to (iii) may be mixed, and then the resulting mixture may be added to the remaining one and mixed. Alternatively, two of (i) to (iii) may be added to the remaining one and mixed. Preferably, (i) and (ii) may be added to (iii) and mixed. Mixing can be performed by inversion or stirring.

[0016] The extracellular vesicle-containing sample (i) can be any sample containing extracellular vesicles. Preferably, the extracellular vesicle-containing sample is a biological liquid sample. The extracellular vesicle-containing sample may be subjected to other treatments before being used in the method of the present invention. Such treatments include, for example, centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

[0017] In one embodiment, the sample containing extracellular vesicles is a culture supernatant sample. The culture supernatant sample may be a cell culture supernatant sample or a tissue culture supernatant sample. Organisms from which the cultured cells or tissues are derived include, for example, mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; livestock such as cows, pigs, and goats; and working animals such as horses and sheep), birds (e.g., chickens), insects, microorganisms (e.g., bacteria), plants, and fish. Preferably, the organism is a mammal, preferably a human.

[0018] In another embodiment, the extracellular vesicle-containing sample is an animal-derived liquid sample. The animal-derived liquid sample is a bodily fluid sample derived from an organism as described above. Examples of bodily fluid samples include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymph, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Preferably, the bodily fluid is a blood sample, urine, or saliva. Examples of plasma include heparin plasma, citrate plasma, sodium fluoride plasma, and plasma containing ACD (acid-citrate-dextrose) or CPD (citrate phosphate dextrose). In general, recovery of extracellular vesicles is difficult compared to culture supernatants from body fluids (e.g., blood, urine, saliva) that contain larger amounts of proteins (e.g., albumin, lysozyme, lactoferrin, histatin, peroxidase, agglutinin, defensin, immunoglobulin) than from culture supernatants. On the other hand, according to the method of the present invention, the amount of extracellular vesicles recovered from samples containing extracellular vesicles is increased, and extracellular vesicles can be recovered with high efficiency and purity even from such body fluids.

[0019] The particles (ii) can be any particles onto which a substance having affinity for the extracellular vesicle membrane is immobilized. The type of particle is not particularly limited, as long as it can immobilize a substance having affinity for the extracellular vesicle membrane and can be recovered from the mixture obtained in step (a) (e.g., by magnetic manipulation or centrifugation). Examples of particles include microparticles, nanoparticles, microbeads, nanobeads, microspheres, and nanospheres. Examples of particles include inorganic particles (e.g., metal particles, silica particles), organic particles (e.g., polymer particles), and organic-inorganic composite particles. The shape of the particles may be spherical or non-spherical (e.g., ellipsoidal).

[0020] The average primary particle size of the particles is not particularly limited. From the viewpoint of ease of recovery, it may be, for example, 0.001 to 1000 μm, preferably 0.01 to 100 μm, more preferably 0.1 to 10 μm, even more preferably 0.5 to 5 μm, and particularly preferably 1 to 3 μm. The average primary particle size of the particles can be measured by the BET method.

[0021] Preferably, the particles are magnetic particles from the viewpoint of ease of recovery by magnetic manipulation. The magnetic particles are particles containing a magnetic material such as iron, nickel, cobalt, or an alloy thereof (e.g., ferrite). The magnetic particles preferably have an average primary particle size of 1 to 3 μm.

[0022] The substance immobilized on the particle and having affinity for the extracellular vesicle membrane is a substance capable of binding to a surface marker of the extracellular vesicle. Examples of surface markers of the extracellular vesicle include tetraspanin membrane proteins (EV membrane-specific four-spanning membrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloproteinase inducers (e.g., CD147), heat shock protein (HSP) 70, HSP90, major histocompatibility complex (MHC) I, lysosome-associated membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, Annexins, Caveolin-I, and EpCAM. The surface marker of the extracellular vesicle is preferably a tetraspanin membrane protein (e.g., CD9, CD63) or an extracellular matrix metalloproteinase inducer (e.g., CD81 or CD147).

[0023] Examples of substances having affinity for the extracellular vesicle membrane include antibodies, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins that are specific to the surface markers of extracellular vesicles. In the present invention, a single substance or multiple (e.g., two, three, or four) types of substances can be used as the substance having affinity for the surface markers of extracellular vesicles.

[0024] Preferably, the substance having affinity for the extracellular vesicle membrane may be an antibody against a surface marker of the extracellular vesicle membrane, from the viewpoints of ensuring specificity for the surface marker of the extracellular vesicle and ease of preparation. Examples of antibodies include full-length antibodies (e.g., monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof. The antigen-binding fragment may be any antibody fragment that maintains binding to the target EV surface marker, such as Fab, Fab', F(ab')2, scFv, etc. In the present invention, a single antibody or multiple types (e.g., two, three, or four types) of antibodies may be used.

[0025] Although the polymer (iii) itself may be used, it is preferable to use an aqueous polymer solution in which the polymer is dissolved in an aqueous solution (e.g., a buffer solution) to reduce viscosity and facilitate mixing. The polymer concentration in the aqueous polymer solution varies depending on factors such as the type of polymer and degree of polymerization, but may be, for example, 0.01 to 30 wt %, preferably 0.02 to 25 wt %, more preferably 0.05 to 20 wt %, even more preferably 0.1 to 15 wt %, and particularly preferably 0.2 to 10 wt %. Preferably, the polymer may be a water-soluble polymer. A water-soluble polymer refers to a polymer whose solubility in water at any temperature between 4 and 80°C (preferably between 4 and 37°C) is 0.01 wt % or more, more preferably 0.1 wt % or more. The solubility of the water-soluble polymer in water at 4 to 80°C (preferably between 4 and 37°C) may be preferably 0.05 wt % or more, more preferably 0.1 wt % or more.

[0026] To achieve the objects of the present invention, the polymer may have a viscosity of 1.5 mPa·s or more in a 1 to 20 wt % aqueous solution at 20 to 30°C. The viscosity of the polymer under the above conditions may be preferably 5 mPa·s or more, more preferably 10 mPa·s or more, even more preferably 20 mPa·s or more, even more preferably 30 mPa·s or more, and particularly preferably 35 mPa·s or more. The viscosity of the polymer under the above conditions may be preferably 30,000 mPa·s or less, more preferably 20,000 mPa·s or less, even more preferably 10,000 mPa·s or less, even more preferably 5,000 mPa·s or less, and particularly preferably 1,000 mPa·s or less. More specifically, the viscosity of the polymer under the above conditions may be preferably 5 to 30,000 mPa·s, more preferably 10 to 20,000 mPa·s, even more preferably 20 to 10,000 mPa·s, even more preferably 30 to 5,000 mPa·s, and particularly preferably 35 to 1,000 mPa·s.

[0027] To achieve the objects of the present invention, the polymer may have a viscosity of 1.5 mPa·s or more at 30°C in a phosphate-buffered saline (PBS) solution prepared by dissolving the polymer in PBS to a concentration of 2% by weight. The viscosity of the polymer under the above conditions may be preferably 5 mPa·s or more, more preferably 10 mPa·s or more, even more preferably 20 mPa·s or more, even more preferably 30 mPa·s or more, and particularly preferably 35 mPa·s or more. The viscosity of the polymer under the above conditions may be preferably 30,000 mPa·s or less, more preferably 20,000 mPa·s or less, even more preferably 10,000 mPa·s or less, even more preferably 5,000 mPa·s or less, and particularly preferably 1,000 mPa·s or less. More specifically, the viscosity of the polymer under the above conditions may be preferably 5 to 30,000 mPa·s, more preferably 10 to 20,000 mPa·s, even more preferably 20 to 10,000 mPa·s, even more preferably 30 to 5,000 mPa·s, and particularly preferably 35 to 1,000 mPa·s.

[0028] The viscosity of a polymer can be measured, for example, by detecting the viscosity friction torque of the liquid generated on the outer periphery of a rotor when a liquid sample is rotated by the rotor (a rotational viscometer method), or by measuring the time it takes for a weight to fall freely through a measuring tube filled with the sample (a falling-ball viscometer method). Alternatively, the viscosity of a polymer can be measured, for example, by placing a vibrating body (viscosity sensor) in a liquid sample and vibrating it. The vibration amplitude of the sensor is suppressed and reduced by the viscous resistance as the liquid viscosity increases. However, the vibration amplitude of the sensor is suppressed and reduced by the viscous resistance of the liquid. The input current is increased to overcome this suppressing force and maintain a constant amplitude, and the amount of input current is measured (a vibration viscometer method). Preferably, the viscosity of a polymer can be measured using a viscosity analyzer (e.g., a rheology spectrometer SKR100, Yamato Scientific Co., Ltd.).

[0029] Furthermore, to achieve the objectives of the present invention, the polymer may have a weight-average molecular weight of, for example, 10 kDa or more. The weight-average molecular weight of the polymer may be preferably 12 kDa or more, more preferably 14 kDa or more, even more preferably 16 kDa or more, even more preferably 18 kDa or more, and particularly preferably 20 kDa or more. The weight-average molecular weight of the polymer may be preferably 5,000 kDa or less, more preferably 3,000 kDa or less, even more preferably 2,000 kDa or less, even more preferably 1,000 kDa or less, and particularly preferably 500 kDa or less. More specifically, the weight-average molecular weight of the polymer may be preferably 12 to 5,000 kDa, more preferably 14 to 3,000 kDa, even more preferably 16 to 2,000 kDa, even more preferably 18 to 1,000 kDa, and particularly preferably 20 to 500 kDa.

[0030] Any polymer having the above-mentioned properties can be used as the polymer, including, for example, polysaccharides, proteins, polyether compounds, and polyvinyl derivatives having hydrophilic groups.

[0031] Polysaccharides are sugar polymers. Examples of polysaccharides include simple polysaccharides (e.g., cellulose, cellulose derivatives, starch, glycogen) and complex polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, heparin). From the viewpoints of improving the recovery efficiency of extracellular vesicles, improving water solubility, and ease of availability, the polysaccharide is preferably a cellulose derivative.

[0032] The cellulose derivative is a cellulose derivative in which the hydrogen atom of at least one hydroxyl group of cellulose is substituted with a hydrophilic group. Examples of the hydrophilic group in the cellulose derivative include carboxyalkyl (e.g., carboxy C 1~6 alkyl), hydroxyalkyl (e.g., hydroxy C 1~6 The hydrophilic group in the cellulose derivative is preferably a carboxyalkyl or hydroxyalkyl group. Examples of carboxyalkyl include carboxymethyl, carboxyethyl (1-carboxyethyl, 2-carboxyethyl), carboxypropyl (1-carboxypropyl, 2-carboxypropyl, 3-carboxypropyl), carboxyisopropyl (1-carboxy-2-methylethyl, 2-carboxy-2-methylethyl), carboxybutyl (1-carboxybutyl, 2-carboxybutyl, 3-carboxybutyl, 4-carboxybutyl), carboxy t-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl, 3-carboxypentyl, 4-carboxypentyl, 5-carboxypentyl), and carboxyhexyl (1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl, 5-carboxyhexyl, 6-carboxyhexyl). Examples of hydroxyalkyl include hydroxymethyl, hydroxyethyl (1-hydroxyethyl, 2-hydroxyethyl), hydroxypropyl (1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl), hydroxyisopropyl (1-hydroxy-2-methylethyl, 2-hydroxy-2-methylethyl), hydroxybutyl (1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl), hydroxy t-butyl, hydroxypentyl (1-hydroxypentyl, 2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl, 5-hydroxypentyl), and hydroxyhexyl (1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl, 5-hydroxyhexyl, 6-hydroxyhexyl). Specific examples of cellulose derivatives include carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), and hydroxypropylmethyl cellulose (HPMC). Cellulose derivatives also include nanocellulose derivatives. Nanocellulose derivatives are derivatives of nanocellulose. Nanocellulose is fibrous cellulose with a fiber width on the order of nanometers. The fiber width of nanocellulose may be, for example, 500 nm or less, preferably 200 nm or less, more preferably 100 nm or less, even more preferably 50 nm or less, even more preferably 10 nm or less, and particularly preferably 5 nm or less.

[0033] Proteins are amino acid polymers also known as polypeptides, and examples of proteins include gelatin, casein, albumin, collagen, and alginic acid.

[0034] A polyether compound is a polymer containing an ether structure in the main chain of a repeating unit. Examples of polyether compounds include polyalkyleneoxy compounds (e.g., polyC 1~6Examples of polyalkyleneoxy compounds include polyethylene glycol and polypropylene glycol. The polyalkyleneoxy compound is preferably polyethylene glycol.

[0035] A polyvinyl derivative having a hydrophilic group is a polyvinyl derivative in which at least one hydrogen atom is substituted with a hydrophilic group, and a polyvinyl derivative in which one hydrogen atom in a methylene unit is substituted with a hydrophilic group is preferred. Examples of the hydrophilic group of the polyvinyl derivative include a carbonyl-containing hydrophilic group, a carboxy-containing hydrophilic group, a nitrogen-containing hydrophilic group, and a ring (carbon ring or heterocyclic ring)-containing hydrophilic group. The hydrophilic group of the polyvinyl derivative is preferably a carbonyl-containing hydrophilic group, a nitrogen-containing hydrophilic group, or a heterocyclic ring-containing hydrophilic group, and more preferably a lactam (e.g., α-lactam, β-lactam, γ-lactam, δ-lactam, ε-lactam). A specific example of a polyvinyl derivative having a hydrophilic group is polyvinylpyrrolidone.

[0036] Polymers such as polysaccharides, proteins, polyvinyl derivatives having hydrophilic groups, and polyether compounds also include their salts, such as salts of metals (e.g., monovalent metals such as lithium, sodium, potassium, rubidium, and cesium, and divalent metals such as calcium, magnesium, and zinc) and salts of inorganic bases (e.g., ammonia).

[0037] Preferably, the polymer is a polysaccharide, a protein, or a polyether compound, more preferably a polysaccharide or a protein, even more preferably a polysaccharide, and particularly preferably a cellulose derivative.

[0038] The concentration of the polymer in the mixture is not particularly limited as long as it allows for more efficient recovery of extracellular vesicles than when the polymer is not contained and the polymer is soluble in the mixture. Such a concentration varies depending on the type of polymer, but may be, for example, 0.01 to 10.00 wt %, preferably 0.05 to 7.50 wt %, and more preferably 0.10 to 5.00 wt %.

[0039] The mixing in step (a) is carried out under conditions sufficient to produce target particles bound to extracellular vesicles via a substance having affinity for the extracellular vesicle membrane. Such temperature conditions are, for example, 4 to 60°C, preferably 15 to 50°C, and more preferably 20 to 45°C. The time required to prepare the mixture is, for example, 30 seconds or less, preferably 20 seconds or less, and more preferably 15 seconds or less. By mixing under such conditions, the amount of target particles bound to extracellular vesicles can be increased.

[0040] The method of the present invention may further comprise incubating the mixture after the mixing in step (a) above. The incubation time varies depending on factors such as the time required to prepare the mixture, the desired amount of extracellular vesicles recovered, and the incubation temperature, but is, for example, 48 hours or less, preferably 24 hours or less, more preferably 120 minutes or less, and even more preferably 60 minutes or less. From the viewpoint of rapid processing, the incubation time is even more preferably 30 minutes or less, and particularly preferably 20 minutes or less, 10 minutes or less, or 5 minutes or less. The incubation temperature is the same as the temperature conditions for the above-mentioned mixing.

[0041] In the step (b), target particles are separated from the mixture obtained in the step (a). The target particles are bound to extracellular vesicles via a substance having affinity for the extracellular vesicle membrane. Therefore, by separating the target particles, extracellular vesicles bound to the target particles can be separated.

[0042] The target particles can be separated from the mixture by any method. This separation can achieve so-called B (bound) / F (free) separation, which separates factors immobilized on the solid phase (target particles) from factors not immobilized on the solid phase (target particles). For example, the target particles can be separated from the mixture by centrifuging the mixture obtained in step (a) and then removing the supernatant. Furthermore, when magnetic particles are used in step (a), the target particles can be separated from the mixture obtained in step (a) by recovering them using a magnetic collection operation.

[0043] In a preferred embodiment, the method of the present invention may further include reducing the viscosity of the mixture obtained in the above step (a). That is, in this embodiment, the method of the present invention may include the following steps (a) to (c): (a) mixing (i) a sample containing extracellular vesicles, (ii) particles having immobilized thereon a substance having affinity for extracellular vesicle membranes, and (iii) a polymer to obtain a mixture containing (i') target particles bound to extracellular vesicles via the substance, and (ii') a polymer; (b) reducing the viscosity of the mixture; and (c) separating the target particles from the solution obtained in (b). Steps (a) and (c) in this embodiment can be carried out in the same manner as steps (a) and (b) in the above-described method, respectively.

[0044] In the preferred embodiment, step (b) can be carried out by any method capable of reducing the viscosity of the mixture obtained in step (a). From the viewpoint of ease of implementation, step (b) is preferably carried out by ultrasonic treatment or treatment with an enzyme having polymer-degrading ability.

[0045] Ultrasonic treatment can be carried out by either a dry method or a water bath method. Dry ultrasonic treatment is a direct ultrasonic treatment that can be achieved by directly or indirectly contacting the ultrasonic generator with a container (e.g., a tube) containing the target solution (e.g., the mixed solution obtained in (a) in the present invention) without using a water bath or the like. Water bath ultrasonic treatment is an indirect ultrasonic treatment that can be achieved by immersing a container (e.g., a tube) containing the target solution in the water bath of an ultrasonic generator. The ultrasonic treatment time is not particularly limited as long as it can reduce the viscosity of the mixed solution, and varies depending on factors such as ultrasonic conditions, but is, for example, 10 seconds to 30 minutes, preferably 20 seconds to 20 minutes, and more preferably 30 seconds to 10 minutes.

[0046] The frequency (Hz) and power (w) of the ultrasonic waves are not particularly limited as long as they can reduce the viscosity of the mixed solution and, in turn, facilitate the separation of target particles from the solution obtained in step (b) in step (c). Such a frequency is, for example, 5 kHz to 5.0 MHz. From the viewpoint of adopting a frequency range that is widely available for any ultrasonic wave generating oscillator or generator, the frequency may be preferably 10 kHz to 3.0 MHz, more preferably 20 kHz to 2.5 MHz, and even more preferably 30 kHz to 2.0 MHz. The power may be, for example, 5 to 300 w. From the same viewpoint as the frequency, the power may be preferably 10 to 200 w.

[0047] In certain embodiments, the ultrasonic frequency (Hz) may be set to significantly improve the recovery efficiency of extracellular vesicles bound to target particles. Such a frequency may be, for example, in the range of 40 kHz or less (preferably 10 kHz to 40 kHz, more preferably 20 kHz to 40 kHz, and even more preferably 30 kHz to 40 kHz) or in the range of 950 kHz or more (preferably 950 kHz to 3.0 MHz, more preferably 950 kHz to 2.5 MHz, and even more preferably 950 kHz to 2.0 MHz).

[0048] The treatment with an enzyme capable of decomposing a polymer can be carried out by appropriately selecting and using an enzyme capable of decomposing the polymer depending on the type of polymer used in the above step (a). For example, if the polymer used in (a) is a polysaccharide such as a cellulose derivative, a glycolytic enzyme can be used. Examples of glycolytic enzymes include cellulase, glycosidase, xylanase, lactase, amylase, chitinase, sucrase, maltase, neuraminidase, invertase, hyaluronidase, and lysozyme. Furthermore, an endo- or exo-enzyme can be used as the glycolytic enzyme. From the viewpoint of efficiently cleaving the polymer in a short time and thereby efficiently reducing the viscosity, the glycolytic enzyme is preferably an endo-enzyme. The glycolytic enzyme can be selected depending on the type of polysaccharide. For example, if the polymer used in (a) is a cellulose derivative, a glycolytic enzyme that decomposes cellulose derivatives, such as cellulase or endoglucanase, can be used. Furthermore, if the polymer used in (b) is a protein, a protease can be used. Examples of protease include aspartic acid proteases, serine proteases, cysteine ​​proteases, and metalloproteases. Furthermore, endoproteases (e.g., trypsin, chymotrypsin, elastase, collagenase) or exoproteases (e.g., leucine aminopeptidase, carboxypeptidase, dipeptidyl aminopeptidase) can be used as the protease. From the viewpoint of efficiently cleaving the polymer in a short time and thereby efficiently reducing the viscosity, the protease may preferably be an endoprotease. When the substance having affinity for the extracellular vesicle membrane used in the present invention is a substance having the ability to bind to a surface marker protein of the extracellular vesicle, from the viewpoint of preventing degradation of the surface marker protein, the enzyme having the ability to decompose polymers is preferably a glycolytic enzyme. Conditions for the treatment with an enzyme having polymer-decomposing ability may be, for example, conditions for a typical enzyme reaction (eg, 25 to 40° C. for 1 to 60 minutes).

[0049] The polymer used in the present invention may be appropriately selected depending on the type of treatment for reducing the viscosity of the mixed solution. For example, when the treatment for reducing the viscosity of the mixed solution is ultrasonic treatment, the polymer can be appropriately selected from, for example, polysaccharides, proteins, polyvinyl derivatives having hydrophilic groups, or polyether compounds. On the other hand, when the treatment for reducing the viscosity of the mixed solution is treatment with an enzyme capable of decomposing polymers, the polymer is preferably a polysaccharide (preferably a cellulose derivative) or a protein, from the viewpoint of ease of obtaining such an enzyme. When the substance having affinity for extracellular vesicle membranes used in the present invention is a substance capable of binding to surface marker proteins of extracellular vesicles, the polymer is preferably a polysaccharide, from the viewpoint of preventing degradation of the surface marker proteins.

[0050] In the method of the present invention, a chelating agent may be used in combination in the mixing step (a) to improve the recovery rate of extracellular vesicles. Treating a sample containing extracellular vesicles with a chelating agent can improve the recovery rate of extracellular vesicles (e.g., International Publication No. 2018 / 070479).

[0051] A chelating agent is a compound or salt thereof having a coordinating moiety capable of forming a coordinate bond with a metal ion. The number of coordinating moieties is preferably two or more, more preferably three or more (e.g., three or six). Examples of the coordinating atom as the coordinating moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordinating atom is preferably an oxygen atom or a phosphorus atom, more preferably an oxygen atom. Examples of the coordinating group as the coordinating moiety include groups having the above-mentioned coordinating atoms. The coordinating group is preferably a carboxylic acid group or a phosphate group, more preferably a carboxylic acid group.

[0052] Examples of chelating agents include hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), glycoletherdiaminetetraacetic acid (EGTA), and salts thereof. Examples of salts include metal salts (e.g., monovalent metal salts such as sodium salts and potassium salts, and divalent metal salts such as calcium salts and 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 alkyl groups), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid).

[0053] The chelating agent may also be a chelating agent commonly used as a component contained in blood collection tubes for clinical tests. Examples of such chelating agents include EDTA, EGTA, NTA, HEDTA, EDTMP, HIDA, citric acid, and salts thereof. In the present invention, the use of such chelating agents is also desirable from the viewpoint of clinical application.

[0054] In the present invention, one type of chelating agent may be used alone, or multiple types (e.g., two, three, or four) of chelating agents may be used in combination. The concentration of the chelating agent varies depending on factors such as the type and concentration of other components used in combination with the chelating agent, but is, for example, 10 mM to 1000 mM.

[0055] The method of the present invention may further include, after separation of the target particles bound to extracellular vesicles via a substance having affinity for the extracellular vesicle membrane, (I) washing the target particles and / or (II) releasing the extracellular vesicles from the target particles.

[0056] The target particles can be washed using an aqueous solution (e.g., a buffer solution), and the number of washing steps is usually 1 to 3 times.

[0057] The release of extracellular vesicles from target particles can be carried out by any method capable of dissociating the bond between the extracellular vesicles and a substance having affinity for the extracellular vesicle membrane, such as treatment with an acid or alkali, or heat treatment.

[0058] 2. Methods for analyzing extracellular vesicles The present invention also provides a method for analyzing extracellular vesicles, including the following (1) and (2): (1) isolating extracellular vesicles from an extracellular vesicle-containing sample; and (2) To analyze isolated extracellular vesicles.

[0059] Step (1) in the analytical method of the present invention can be carried out in the same manner as in the method for recovering extracellular vesicles of the present invention.

[0060] In the above step (2), examples of the analysis targets in the analysis of extracellular vesicles include components contained in extracellular vesicles (e.g., components contained inside extracellular vesicles, membrane components of extracellular vesicles, and components present on the membrane surface of extracellular vesicles), and the extracellular vesicles themselves.

[0061] Analysis of components contained in extracellular vesicles can be performed qualitatively or quantitatively. Such analysis can also be of one component or multiple components. Components that can be analyzed include, for example, proteins, nucleic acids (e.g., RNA, DNA), sugars, lipids, amino acids, vitamins, polyamines, and peptides. According to the present invention, the recovery amount of extracellular vesicles is increased, allowing the components in extracellular vesicles to be analyzed with high accuracy.

[0062] The analysis of the components can be carried out by any method. When the component to be analyzed is a protein, analytical methods include, for example, immunoassays and mass spectrometry. Immunoassays include, for example, direct competitive assays, indirect competitive assays, and sandwich assays. Furthermore, such immunoassays include chemiluminescent immunoassays (CLIA) (e.g., chemiluminescent enzyme immunoassays (CLEIA)), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), immunochromatography, Western blotting, immunostaining, and fluorescence activated cell sorting (FACS). When multiple components are analyzed, proteome analysis may be performed. When the component to be analyzed is a nucleic acid, analytical methods include, for example, hybridization methods using probes, gene amplification methods using primers (eg, 2, 3, or 4 primers), and mass spectrometry. When the components to be analyzed are components other than proteins and nucleic acids, analytical methods include, for example, immunoassays and mass spectrometry. When multiple components are analyzed, metabolomic analysis may be performed.

[0063] Analysis of extracellular vesicles themselves can also be performed qualitatively or quantitatively. For example, analysis of extracellular vesicles can be performed using instruments such as particle analysis instruments, electron microscopes, and flow cytometers. In this case, the number of extracellular vesicles, the size and shape of the particles, and their distribution can be analyzed.

[0064] It has been reported that extracellular vesicles may be involved in various diseases such as cancer (WO 2014 / 003053; WO 2014 / 152622; Taylor et al., Gynecologic Oncol, 100 (2008) pp13-21). Therefore, the present invention is useful, for example, for diagnosis and drug discovery based on extracellular vesicles.

[0065] 3. Kit The present invention also provides a kit that can be used in the above-described method of the present invention.

[0066] The kit of the present invention comprises the following (a) to (c): (a) polymer; (b) a substance that has an affinity for the extracellular vesicle membrane; and (c) Enzymes capable of degrading polymers.

[0067] In the kit of the present invention, the substance having affinity for extracellular vesicle membranes is in a free form or immobilized on particles. When the substance having affinity for extracellular vesicle membranes is in a free form, the kit of the present invention may further comprise particles. The kit of the present invention may also further comprise a chelating agent. The definitions, examples, and preferred examples of the polymer, the substance having affinity for extracellular vesicle membranes, the enzyme having polymer-degrading ability, and the chelating agent are the same as those described above in the method of the present invention. The kit of the present invention is useful, for example, for easily and quickly implementing the method of the present invention. [Example]

[0068] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0069] Example 1 We investigated the effects of three commercially available CMC sodium salts (hereafter simply referred to as "CMC"; CAS No. 9004-32-4, Nacalai Tesque #07326-95 (average molecular weight unknown), Sigma-Aldrich #C5678 (average molecular weight 90 kDa), and #C4888 (average molecular weight 250 kDa)) on EV recovery. EV recovery was performed using an anti-CD9 antibody. Serum from healthy donors was centrifuged at 20,000 × g at 4°C for 15 minutes, and 200 μL of serum was diluted with 200 μL of PBS (2.9 mM NaH2PO4, 9.0 mM Na2HPO4, 137 mM NaCl), EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration after dilution of the serum was 50 mM) (ED / EGTA), or CMC-PBS (PBS containing CMC so that the final concentration after dilution of the serum was 0.2–2.5 wt%) to a final concentration of 0.2–2.5 wt%, and magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (manufactured in-house) were added to a final concentration of 0.26 mg / mL. After overnight rotation at 4°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. In Western blotting samples, the exosome-containing sample was treated with SDS, disrupting the exosomes and releasing exosome marker proteins (e.g., CD9) into the sample solution. The immunoprecipitation efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (in-house produced) (Figure 1). All three CMCs demonstrated improved EV recovery efficiency compared to the PBS-diluted sample. The physical properties of the CMC used in the examples are summarized in Table 1 below.

[0070] [Table 1]

[0071] Example 2 The effects of CMC (Sigma-Aldrich #C4888) concentration (final concentration of 0.06 wt% to 1.0 wt%) and reaction temperature (4°C, 37°C) on EV recovery were examined. Serum from healthy volunteers was centrifuged at 20,000 × g for 15 minutes at 4°C. 200 μL of serum was diluted with 200 μL of PBS or CMC-PBS (PBS containing CMC to a final concentration of 0.06–1.0 wt% after serum dilution). Magnetic particles (Dynabeads M-280 tosylactivated, Life Technologies) immobilized with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After overnight incubation at 4°C or 1 hour at 37°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. EV recovery efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (Figure 2). At reaction temperatures of 4°C and 37°C, a final CMC concentration in the range of 0.25% to 1% by weight was found to improve EV recovery efficiency compared to the PBS-diluted sample.

[0072] Example 3 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined using nanoparticle tracking analysis (NanoSight LM10, Quantum Design). 200 μL of healthy human serum was centrifuged at 20,000 × g at 4°C for 15 minutes, and the supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration after serum dilution was 50 mM) (ED / EG), or CMC-PBS (PBS in which CMC was dissolved so that the final concentration after serum dilution was 0.5 wt%) (CMC), and magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After 30 minutes of rotation at 37°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and incubated with 40 μL of Britton & Robinson Universal Buffer (BRUB) (pH 2.6) for 5 minutes. The particles were then neutralized with 20 μL of 1 M Tris-HCl (pH 8.0) to release the extracellular vesicles from the antibody-magnetic particles. Total protein concentration was quantified using a Qubit protein assay (Life Technologies), and 450 μL of PBS was added. The particle count was analyzed using a NanoSight (Figure 3). Diluting with CMC increased the total number of recovered EV particles and the number of particles per total protein, demonstrating the recovery of highly pure extracellular vesicles.

[0073] Example 4 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined in serum and plasma containing five types of anticoagulants (heparin, EDTA, citrate, ACD (acid-citrate-dextrose), and CPD (citrate phosphate-dextrose)). 200 μL of healthy donor serum and anticoagulant-containing healthy donor plasma were centrifuged at 20,000 × g for 15 minutes at 4°C. The supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA to a final concentration of 50 mM after dilution of serum or plasma) (ED / EG), CMC-PBS (PBS containing CMC to a final concentration of 0.5% by weight after dilution of serum or plasma) (CMC), or EDTA / EGTA / CMC-PBS (PBS containing EDTA, EGTA, and CMC to a final concentration of 37.5 mM / 37.5 mM / 0.5% by weight after dilution of serum or plasma) (ED / EG / C). Magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies)) bearing immobilized anti-CD9 antibodies (manufactured in-house) were added to the supernatant at a concentration of 0.26 mg / mL. After a 1-hour rotational reaction at 37°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The immunoprecipitation efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (Figure 4). Regardless of the type of anticoagulant, the effect of CMC on improving EV recovery efficiency was observed even in plasma samples. Furthermore, combining CMC with a chelating agent further improved EV recovery efficiency.

[0074] Example 5 We investigated the effect of CMC (Sigma-Aldrich #C4888) on EV recovery using antibodies against two tetraspanin membrane proteins other than CD9 (CD63 and CD81) and an extracellular matrix metalloproteinase inducer (CD147). 200 μL of healthy human serum was centrifuged at 20,000 × g at 4°C for 15 minutes. The supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (50 mM final concentration after serum dilution) (ED / EG), CMC-PBS (0.5% by weight final concentration after serum dilution) (CMC), or EDTA / EGTA / CMC-PBS (37.5 mM / 37.5 mM / 0.5% by weight final concentration after serum dilution) (ED / EG / C). Antibodies immobilized on magnetic particles (Dynabeads M-280 tosylactivated, Life Technologies) containing anti-CD63 (8A12, Cosmo Bio), anti-CD81 (M38, Abcam), and anti-CD147 (MEM-M6 / 1, Abcam) were added to the supernatant at a concentration of 0.26 mg / mL. After overnight rotation at 4°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. EV collection efficiency was analyzed by Western blotting using an anti-CD63 antibody (in-house), an anti-CD81 antibody (12C4: Cosmo Bio), and a biotinylated anti-CD9 antibody (in-house) (Figures 5A and 5B). The effect of CMC on EV collection efficiency was also confirmed when antibodies against CD63, CD81, and CD147 were used. Furthermore, combining CMC with a chelating agent further improved EV collection efficiency.

[0075] Example 6 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined in two types of body fluids (urine and saliva). Two hundred microliters of urine and saliva samples (200 μL each, designated "#1" and "#2") from healthy volunteers were centrifuged at 15,000 × g for 15 minutes at 4°C and filtered through a 0.22 μm filter. The samples were then diluted with 200 μL of PBS, EDTA / EGTA-PBS (50 mM final concentration after dilution of urine or saliva) (ED / EG), CMC-PBS (0.5% by weight final concentration after dilution of urine or saliva) (CMC), or EDTA / EGTA / CMC-PBS (37.5 mM / 37.5 mM / 0.5% by weight final concentration after dilution of urine or saliva) (ED / EG / C). Magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies)) bearing immobilized anti-CD9 antibodies (manufactured in-house) were added to a final concentration of 0.26 mg / mL. After rotating the mixture at 37°C for 1 hour, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The immunoprecipitation efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (Figure 6). The effect of CMC on improving EV recovery efficiency was confirmed not only in serum and plasma, but also in urine and saliva.

[0076] Example 7 The effects of the cellulose derivatives shown in Table 2 below (final concentrations of 0.13 wt % to 4.0 wt %) on EV recovery were investigated. Serum from healthy volunteers was centrifuged at 20,000 × g for 15 min at 4°C. 200 μL of serum was diluted with 200 μL of PBS, CMC-PBS (final concentration after serum dilution: 0.5 wt%) (CMC), or cellulose derivative dissolved in PBS (final concentrations after serum dilution: 0.13–4.0 wt%). Magnetic particles (Dynabeads M-280 tosylactivated, Life Technologies) immobilized with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After 1 h of rotation at 37°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. EV recovery efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (Figure 7A–C). The three cellulose derivatives other than CMC also showed improved EV recovery efficiency compared to the PBS-diluted sample (HEC in the range of 0.13 wt% to 2.0 wt%, HPC in the range of 0.25 wt% to 4.0 wt%, and HPMC in the range of 0.25 wt% to 2.0 wt%).

[0077] [Table 2]

[0078] Example 8 The effect of polyvinylpyrrolidone (final concentrations of 1 wt%, 2 wt%, and 4 wt%) shown in Table 3 below on EV recovery was examined. Serum from healthy volunteers was centrifuged at 20,000 × g for 15 minutes at 4°C. 200 μL of serum was diluted with 200 μL of PBS or polyvinylpyrrolidone in PBS (final concentrations of 1.0–4.0 wt% after serum dilution). Magnetic particles (Dynabeads M-280 tosylactivated, Life Technologies) immobilized with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After 1 hour of rotation at 37°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. EV recovery efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 8). Polyvinylpyrrolidone concentrations ranging from 1.0 wt% to 4.0 wt% improved EV recovery efficiency compared to the PBS-diluted sample.

[0079] [Table 3]

[0080] Example 9 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery at various reaction temperatures from 35°C to 60°C was examined. Serum from healthy volunteers was centrifuged at 20,000 × g for 15 minutes at 4°C. 100 μL of serum was diluted with 100 μL of PBS or CMC-PBS (final concentration after serum dilution: 0.5 wt%) and magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (in-house production), anti-CD63 antibody (8A12: Cosmo Bio), or anti-CD81 antibody (12C4: Cosmo Bio) were added at a concentration of 0.26 mg / mL. After 5 minutes of incubation at each reaction temperature, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. EV recovery efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (in-house), anti-CD63 antibody (in-house), and anti-CD81 antibody (12C4: Cosmo Bio) (Figures 9A-9C). Addition of CMC was found to improve EV recovery efficiency at reaction temperatures ranging from 35°C to 60°C. Furthermore, at high temperatures above 40°C, addition of CMC was even more effective in improving EV recovery efficiency. Furthermore, the effect of CMC on improving EV recovery efficiency was observed even in short-term reactions.

[0081] Example 10 The viscosity of the cellulose derivative used in Example 7 and the polyvinylpyrrolidone used in Example 8 in a PBS solution was measured. Specifically, each PBS solution was prepared by dissolving each cellulose derivative or polyvinylpyrrolidone in PBS to a concentration of 2 wt %, and the viscosity was calculated as the average of the measurements taken at 30°C, 60 seconds for a measurement time, and rotation speeds of 200 rpm, 400 rpm, 600 rpm, and 800 rpm (Table 4).

[0082] [Table 4]

[0083] Example 11 We investigated the effect of immunoprecipitation using antibodies against CMC (Sigma-Aldrich #C4888) and tetraspanin membrane proteins (CD9 and CD63) on EV recovery and on the detection of marker (EML4-ALK fusion gene) RNA from EVs. The culture supernatant of human lung cancer cells H2228 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000 × g at 4°C for 5 minutes, filtered through a 0.22 μm filter (Millipore), and then concentrated 100-fold using an Amicon Ultra-15 (Millipore). The concentrate was diluted with an equal volume of PBS, EDTA / EGTA-PBS (50 mM final concentration after dilution of the concentrate) (ED / EG), CMC-PBS (0.5% by weight final concentration after dilution of the concentrate) (CMC), or EDTA / EGTA / CMC-PBS (37.5 mM / 37.5 mM / 0.5% by weight final concentration after dilution of the concentrate) (ED / EG / C). Anti-CD9 antibody (in-house production) and anti-CD63 antibody (in-house production)-immobilized Dynabeads M-280 tosylactivated (Life Technologies) were added to each solution at 0.26 mg / mL. After overnight rotation at 4°C, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated magnetic particles were washed three times with PBS-T, and total RNA was purified using the miRNeasy micro kit (QIAGEN). cDNA was prepared from the purified total RNA using the SuperScript™ IV First-Strand Synthesis System (Thermo Fisher Scientific), and EML4-ALK mRNA was quantified using Droplet Digital PCR (BioRad) (Figure 10). Primers and a fluorescent probe with the sequences shown in Table 5 were used to detect EML4-ALK mRNA. The fluorescent probe used was a double-quencher probe with the fluorescent substance HEX at the 5' end, a ZEN quencher inside the probe, and an Iowa Black (registered trademark) quencher (IABkFQ) at the 3' end. Using the primers and fluorescent probe with the sequences shown in Table 5, variants 3a and 3b of the EML4-ALK fusion gene can be detected.

[0084] [Table 5]

[0085] EML4-ALK was detected in EVs collected by immunoprecipitation. The use of CMC increased the amount of EML4-ALK mRNA detected, demonstrating an improved EV collection efficiency. Furthermore, the addition of a chelating agent further increased the amount of EML4-ALK mRNA detected, demonstrating an improved EV collection efficiency.

[0086] Example 12 Study on the effect of ultrasound on the collection efficiency of magnetic particles (1) The effect of ultrasound on the magnetic particle collection efficiency was investigated by reacting extracellular vesicles (EVs) and antibody-bound magnetic particles in the presence of carboxymethylcellulose (CMC) and then irradiating them with ultrasound (water bath).

[0087] Anti-CD9 antibody-immobilized magnetic particles (Dynabeads M-280 tosylactivated (Life Technologies #14204)) at a final concentration of 1.2 mg / mL and 35.4 ng of EVs recovered from DU145 (a human prostate cancer cell line) were added to 600 μL of EDTA / EGTA / CMC-PBS (final concentrations of 50 mM / 50 mM / 0.5 wt%) in a 2 mL tube. The mixture was incubated at 37°C for 1 hour to allow binding. The tube was then irradiated for 3 minutes at frequencies of 100 kHz (50 watts), 200 kHz (100 watts), 950 kHz (100 watts), and 1.6 MHz (100 watts) using a water bath ultrasonicator (Kaijo QUAVAmini QR-001 and QR-003). A non-irradiated tube was also prepared as a negative control. Instead of ultrasonic irradiation, cellulase (Cellulase from Aspergillus sp. SIGMA #C2605-50ML) was added in an amount of 1 / 150 of the solution, and the solution was allowed to react at 37°C for 5 minutes. A water bath-type ultrasonic vibrator was used. After ultrasonic irradiation under each condition, magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation), and the separated solution was recovered. The degree of magnetic particle contamination was evaluated using measurements taken with a spectrophotometer (wavelength 500 nm) (JASCO Corporation V-650). The CMC used had an average molecular weight of 250 kDa #C4888 (Sigma-Aldrich).

[0088] As a result, when irradiation was performed at 950 kHz or higher, the amount of magnetic particles mixed into the solution after B / F separation was significantly reduced compared to when irradiation was not performed. This indicates that irradiation at 950 kHz or higher significantly improved the magnetic collection efficiency, demonstrating a high magnetic collection efficiency (Figure 11). Furthermore, the addition of cellulase also demonstrated a similarly high magnetic collection efficiency.

[0089] Example 13 Study on the effect of ultrasound on the collection efficiency of magnetic particles (2) We investigated the effect of improving the collection efficiency of ultrasound on EV recovery by reacting EVs and antibody-bound particles in the presence of CMC and then irradiating them with ultrasound.

[0090] Dynabeads M-280 tosylactivated (Life Technologies #14204) final solution (1.2 mg / mL) immobilized with anti-CD9 antibody and 1.3 μg of EV recovered from BxPC3 (a human pancreatic adenocarcinoma cell line) were added to 600 μL of EDTA / EGTA / CMC-PBS (final concentrations of 50 mM / 50 mM / 0.5 wt%) in a 2 mL tube. The mixture was incubated at 37°C for 1 hour to allow binding, and then irradiated for 3 minutes at a frequency of 30 kHz (35 watts) (Kaijo QUAVAmini QR-001). A negative control was prepared without irradiation. A dry ultrasonic transducer was used. After ultrasonic irradiation, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation), and the separated solution was recovered (recovered supernatant). The amount of magnetic particles in the recovered supernatant was evaluated by comparing the measurements taken with a spectrophotometer (wavelength 500 nm) (JASCO Corporation V-650) (Figure 12A). Furthermore, the amount of CD9 contained in the collected solution was evaluated by sandwich CLEIA using an anti-CD9 antibody (Fig. 12B). Furthermore, the EV recovery efficiency of the magnetic particles after B / F separation was analyzed by Western blotting with anti-CD9 antibody (25 kDa) (Figure 12C). Specifically, the sandwich CLEIA using the anti-CD9 antibody was performed as follows. First, Dynabeads immobilized with anti-CD9 antibody were mixed with the collected solution, and the resulting mixture was incubated at 37°C for 8 minutes. After B / F separation and washing, 50 μL of alkaline phosphatase-labeled anti-CD9 antibody was added, stirred, and incubated at 37°C for 8 minutes, followed by B / F separation and washing. Next, 200 μL of Lumipulse® substrate solution (Fujirebio Inc.) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt was dispensed. After stirring, the mixture was incubated at 37°C for 4 minutes, and the luminescence was measured using a luminometer. Count values ​​were obtained as the measured values. The luminescence intensity was measured using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (Fujirebio)). The lower the amount of CD9 contained in the collected solution (the lower the CD9 count value), the higher the EV collection efficiency.

[0091] As a result, it was found that the ultrasonically irradiated sample had improved magnetic collection efficiency and EV recovery efficiency compared to the sample without irradiation (Figures 12A to 12C).

[0092] Example 14 Investigating the effect of CMC concentration on the magnetic collection efficiency of magnetic particles The effect of CMC concentration on the magnetic collection efficiency of magnetic particles was investigated.

[0093] Three CMC concentrations (1 wt%, 0.5 wt%, and 0.25 wt%) were added to 600 μL of EDTA / EGTA / CMC-PBS (final concentrations: 50 mM / 50 mM / 1 wt%, 0.5 wt%, and 0.25 wt%). Anti-CD9 antibody-immobilized Dynabeads M-280 tosylactivated (Life Technologies #14204) final 1.2 mg / mL and 1.3 μg of EVs recovered from BxPC3 (a human pancreatic adenocarcinoma cell line) were added. The mixture was incubated at 37°C for 1 hour to allow binding. The mixture was then irradiated for 3 minutes at 30 kHz (35 watts) using a Kaijo QUAVAmini QR-001. A negative control was prepared using EDTA / EGTA / CMC-PBS solution containing 0.5 wt% CMC without ultrasound. A dry ultrasonic transducer was used. After ultrasonic irradiation, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation), and the separated solution was recovered. The amount of magnetic particle contamination was compared using measurements taken with a spectrophotometer (wavelength 500 nm) (JASCO Corporation V-650).

[0094] As a result, in the EDTA / EGTA / CMC solution in which the CMC concentration was set to 0.5 wt % or less, the ultrasonically irradiated sample showed an improvement in magnetic collection efficiency compared to the sample without irradiation (Figure 13).

[0095] Example 15 Examination of the effect of ultrasound on the collection efficiency of magnetic particles in solutions containing cellulose derivatives other than CMC We investigated whether ultrasound could improve the magnetic collection efficiency of magnetic particles in solutions containing cellulose derivatives other than CMC.

[0096] To 600 μL of a cellulose derivative solution (0.25 wt%-4.0 wt%) in PBS, 1.2 mg / mL of Dynabeads M-280 tosylactivated (Life Technologies #14204) anti-CD9 antibody-immobilized final volume and 1.3 μg of EVs recovered from BxPC3 (a human pancreatic adenocarcinoma cell line) were added. The mixture was incubated at 37°C for 1 hour to allow binding, and then irradiated for 3 minutes at a frequency of 30 kHz (35 watts) (Kaijo QUAVAmini QR-001). A negative control was prepared using an EDTA / EGTA / CMC-PBS solution containing 0.5 wt% CMC, without irradiation. A dry ultrasonic transducer was used. After ultrasonic irradiation, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated solution was recovered and the magnetic particle contamination was compared using spectrophotometer measurements (wavelength 500 nm) (JASCO V-650). The cellulose derivatives used were hydroxypropyl cellulose (HPC): average molecular weight 80 kDa #435007, and hydroxyethyl cellulose (HEC): average molecular weight 380 kDa #308633 (both Sigma-Aldrich).

[0097] As a result, it was confirmed that the magnetic collection efficiency of the ultrasonically irradiated samples was improved compared to the samples without ultrasonic irradiation, even in solutions containing cellulose derivatives other than CMC. The effect was particularly notable when the concentration of HPC was 2.0 wt% or less and the concentration of HEC was 0.5 wt% or less (Figure 14).

[0098] Example 16 Investigation of the effect of ultrasonic power on the collection efficiency of magnetic particles We investigated whether the ultrasonic output power affected the magnetic particle collection efficiency.

[0099] Dynabeads M-280 tosylactivated (Life Technologies #14204) anti-CD9 antibody-immobilized (final concentration: 1.2 mg / mL) and 1.3 μg of EVs recovered from BxPC3 (human pancreatic adenocarcinoma cell line) were added to 600 μL of EDTA / EGTA / CMC-PBS (final concentrations: 50 mM / 50 mM / 0.5 wt%) in a 2 mL tube and allowed to bind for 1 hour at 37°C. The cells were irradiated for 3 minutes at 30 kHz frequency and 10, 20, or 35 watts (Kaijo QUAVAmini QR-001). An EMERSON BRANSONIC M1800-J water bath ultrasonicator (40 kHz frequency, 70 watts) was also used instead of the dry ultrasonicator. A non-irradiated negative control was also prepared. After ultrasonic irradiation, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation), and the separated solution was recovered. The amount of magnetic particle contamination was compared using measurements taken with a spectrophotometer (wavelength 500 nm) (JASCO Corporation V-650).

[0100] As a result, it was found that the ultrasonic irradiated sample showed improved magnetic collection efficiency compared to the sample without irradiation under all output conditions (Figure 15).

[0101] Example 17 Examination of the effect of glycolytic enzymes on EV recovery efficiency We investigated whether glycolytic enzymes could improve EV recovery efficiency in a solution containing cellulose derivatives.

[0102] 200 μL of healthy human plasma was centrifuged at 20,000 x g for 15 minutes at 4°C, then diluted with 200 μL of EDTA / EGTA / CMC-PBS (final concentrations of 37.5 mM / 37.5 mM / 0.5 wt%) and anti-CD9 antibody-immobilized Dynabeads M-280 tosylactivated (Life Technologies) at 0.26 mg / mL. After overnight rotation at 4°C, endoglucanase was added to reduce the viscosity of the solution by degrading the CMC. The magnetic particles were then recovered from the solution (B / F separation) and washed three times with PBS-T at 37°C. Next, the sample was prepared in sample buffer (BIO-RAD) for Western blotting, and EV recovery efficiency was analyzed by Western blotting with an anti-CD9 antibody.

[0103] As a result, it was confirmed that glycolytic enzymes improved the EV collection efficiency (Figure 16). It is thought that glycolytic enzymes improved the EV collection efficiency by improving the magnetic collection efficiency of magnetic particles.

[0104] Example 18 Examining the effect of specimen type on EV recovery efficiency We investigated whether the effect of ultrasound on improving magnetic collection efficiency could also be observed in serum samples.

[0105] A 300 μL serum sample, 300 μL of CMC (final concentration 0.5 wt%), and 300 μL of HEC (final concentration 0.25 wt%) were added to a tube, followed by the addition of Dynabeads M-280 tosylactivated (Life Technologies #14204) final 1.2 mg / mL anti-CD9 antibody-immobilized solution. The mixture was rotated overnight at 4°C and then irradiated for 3 minutes at a frequency of 30 kHz (35 watts) (Kaijo QUAVAmini QR-001). A negative control was prepared using CMC (final concentration 0.5 wt%) without irradiation. After ultrasonic irradiation, the magnetic particles were separated from the rest of the solution by magnetic collection (B / F separation). The separated solution was collected, and the magnetic particle contamination was compared using a spectrophotometer (500 nm wavelength) (JASCO V-650) (Table 6, magnetic particle contamination level). After removing the separated solution, the magnetic particles in the tube were washed three times with PBS-T and then divided into two tubes. One tube was incubated with 40 μL of Britton & Robinson Universal Buffer (BRUB) (pH 2.6) for 5 minutes, then neutralized with 20 μL of 1 M Tris-HCl (pH 8.0) to release the extracellular vesicles from the antibody particles. This was then used for nanoparticle tracking analysis (NanoSight LM10, Quantum Design) (Figure 17). The other tube was used for Western blotting analysis using sample buffer (BIO-RAD), and the EV recovery efficiency was analyzed by Western blotting with an anti-CD9 antibody (Figure 18).

[0106] Figure 17 shows the nanoparticle tracking analysis of extracellular vesicles released from magnetic particles, plotting particle diameter on the horizontal axis and the number of particles / mL on the vertical axis. The concentration of extracellular vesicles released from magnetic particles, calculated from the results of Figure 17, is shown in Table 6 (number of particles / mL). As a result, even in serum samples, ultrasonic treatment improved the magnetic collection efficiency and EV recovery efficiency compared to no ultrasonic treatment (Table 6, Figures 17 and 18).

[0107] [Table 6]

Claims

1. A method for recovering extracellular vesicles, comprising the following steps (a) to (c): (a) mixing (i) an extracellular vesicle-containing sample, (ii) particles having immobilized thereon a substance having affinity for the extracellular vesicle membrane, and (iii) a polymer to obtain a mixture containing (i') target particles bound to the extracellular vesicles via the substance, and (ii') a polymer; (b) treating the mixture with an enzyme having polymer-degrading properties or with ultrasound; and (c) separating the target particles from the solution obtained in (b).

2. The method of claim 1, further comprising, after separation of the target particles, (I) washing the target particles and / or (II) releasing extracellular vesicles from the target particles.

3. 3. The method of claim 1 or 2, wherein the ultrasonication is dry or water bath.

4. The method according to any one of claims 1 to 3, wherein the frequency of the ultrasound is in the range of 40 kHz or less or 950 kHz or more.

5. The method according to any one of claims 1 to 4, wherein the polymer is a polysaccharide, a protein, or a polyvinyl derivative having a carbonyl-containing hydrophilic group.

6. 6. The method according to claim 5, wherein the polysaccharide is a cellulose derivative in which the hydrogen atom of at least one hydroxyl group in cellulose is substituted with a carboxyalkyl or hydroxyalkyl group.

7. The method of any one of claims 1 to 6, wherein the polymer has a weight average molecular weight of 10 kDa or more.

8. The method according to any one of claims 1 to 7, wherein the concentration of the polymer in the mixed solution (a) is 0.01 to 10.00% by weight.

9. The method according to any one of claims 1 to 8, wherein (iv) a chelating agent is further mixed in (a).

10. The method according to any one of claims 1 to 9, wherein the substance having an affinity for extracellular vesicle membranes is an antibody against a tetraspanin membrane protein or an antibody against an extracellular matrix metalloproteinase inducer.

11. The method according to any one of claims 1 to 10, wherein the sample containing extracellular vesicles is an animal-derived liquid sample or a culture supernatant sample.

12. A method for analyzing extracellular vesicles, comprising the following (1) and (2): (1) Separating extracellular vesicles from an extracellular vesicle-containing sample by the method according to any one of claims 1 to 11; and (2) Analyzing the isolated extracellular vesicles.

13. (a) a polymer, (b) a substance having an affinity for the extracellular vesicle membrane, and (c) an enzyme having an ability to decompose the polymer; the substance is in free form or immobilized on a particle, If the substance is in free form, the kit may further comprise particles.

14. 14. The kit according to claim 13, wherein the polymer is a polysaccharide or a protein, and the enzyme capable of decomposing the polymer is a glycolytic enzyme or a protease.

Images