Extracellular vesicle purification kit and method for purifying extracellular vesicle

Abstract

To provide an extracellular vesicle purification kit in which the recovery rate of extracellular vesicles is improved.SOLUTION: Provided is an extracellular vesicle purification kit including the following (A) and (B): (A) a solid-phase carrier; and (B) a component containing two or more selected from the group consisting of carbonate ions, phosphate ions, and calcium ions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an extracellular vesicle purification kit and a method for purifying extracellular vesicles. [Background technology]

[0002] In recent years, there has been a demand for a technology to recover vesicles with heterogeneous lipid bilayer structures (hereinafter referred to as "extracellular vesicles") released by cells from samples. Extracellular vesicles are generally purified by centrifugal procedures such as ultracentrifugation or spin columns, but the recovery rate is low. As a product that allows extracellular vesicles to be purified more easily, magnetic particles with antibodies that can specifically recognize extracellular vesicles bound to their surface have been disclosed. However, because these magnetic particles rely on a mechanism that recognizes extracellular vesicles, they are not able to recover all extracellular vesicles, and the problem is that extracellular vesicles are biased toward a specific population.

[0003] As a technique for adsorbing extracellular vesicles, a device in which zinc oxide protrusions are formed in a flow channel has been disclosed (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6606786 Summary of the Invention [Problem to be solved by the invention]

[0005] However, Patent Document 1 does not examine the conditions suitable for recovering extracellular vesicles, and therefore it was unclear what kind of aqueous solution should be used in combination to increase the recovery rate of extracellular vesicles.

[0006] The present invention has been made in view of the above-mentioned problems, and aims to provide an extracellular vesicle purification kit with an improved recovery rate of extracellular vesicles. Another aim of the present invention is to provide a method for purifying extracellular vesicles using the above-mentioned extracellular vesicle purification kit. [Means for solving the problem]

[0007] In view of the above, the present inventors have conducted extensive research and have found that the above problems can be solved by combining a solid phase carrier with a specific salt, thereby completing the present invention.

[0008] Each aspect of the present invention is described below. [1] An extracellular vesicle purification kit comprising the following (A) and (B): (A) Solid support. (B) A component containing two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions. [2] The extracellular vesicle purification kit according to [1], further comprising the following (C): (C) Chelating agent. [3] The extracellular vesicle purification kit according to [1] or [2], wherein the component (B) is a component containing carbonate ions, phosphate ions, and calcium ions. [4] The kit for extracellular vesicle purification according to any one of [1] to [3], wherein the component (B) is a component containing a carbonate, a phosphate, and a calcium salt. [5] The extracellular vesicle purification kit according to [4], wherein the carbonate is sodium bicarbonate, the phosphate is sodium dihydrogen phosphate or disodium hydrogen phosphate, and the calcium salt is calcium chloride. [6] The kit for extracellular vesicle purification according to any one of [1] to [5], wherein the solid phase carrier has a metal oxide on the surface thereof. [7] The extracellular vesicle purification kit described in [6], wherein the metal oxide is a single or multiple component selected from the group consisting of zinc oxide, titanium oxide, silicon dioxide, nickel oxide, yttria oxide, tin oxide, indium oxide, and indium tin oxide. [8] The kit for purifying extracellular vesicles according to any one of [1] to [7], wherein the solid phase carrier is a magnetic particle having a metal oxide on the surface. [9] The kit for extracellular vesicle purification according to any one of [1] to [8], wherein the solid phase carrier has a non-adhesive layer for extracellular vesicles on its surface.

[10] A method for purifying extracellular vesicles using the extracellular vesicle purification kit described in any one of [1] to [9], comprising: an adsorption step of mixing a liquid containing the extracellular vesicles with the component (A) and the component (B) and adsorbing the extracellular vesicles to the surface of the component (A); a separation step of separating the component (A) to which the extracellular vesicles have been adsorbed from the liquid; a washing step of washing the component (A) to which the extracellular vesicles have been adsorbed; and a desorption step of detaching the extracellular vesicles from the component (A) to which the extracellular vesicles have been adsorbed. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide an extracellular vesicle purification kit with an improved recovery rate of extracellular vesicles. According to the present invention, it is possible to provide a method for purifying extracellular vesicles using the above-mentioned extracellular vesicle purification kit. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an example of a solid phase carrier according to one embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram showing an example of a solid phase carrier according to another embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, a mode for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The following present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out by appropriately modifying it within the scope of its spirit.

[0012] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. Furthermore, unless specifically stated otherwise, the units of the numerical values ​​before and after "to" are the same. In the numerical ranges described in stages in this specification, the upper or lower limit of a numerical range in a certain stage may be replaced with the upper or lower limit of a numerical range in another stage. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in the examples. Furthermore, the upper and lower limits individually described can be combined in any way. In this specification, "(meth)acrylate" means at least one of acrylate and the corresponding methacrylate.

[0013] [Extracellular vesicle purification kit] The term "extracellular vesicle purification kit" as used herein refers to a kit used to separate extracellular vesicles from a sample containing extracellular vesicles. Extracellular vesicles are vesicles with a heterogeneous lipid bilayer structure released by cells. Extracellular vesicles may encapsulate microRNA (microribonucleic acid; hereinafter, sometimes referred to as "miRNA") and CD63 (protein), which function as early diagnostic markers for cancer and other conditions. Examples of samples containing extracellular vesicles include cell culture media (culture supernatants), blood, serum, plasma, urine, sweat, saliva, and breast milk.

[0014] The extracellular vesicle purification kit according to this embodiment comprises the following (A) and (B): (A) A solid phase carrier (hereinafter also referred to as "component (A)"). (B) a component containing two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions (hereinafter also referred to as "component (B)").

[0015] [Component (A)] The shape of the solid phase carrier can be selected appropriately depending on the application, such as particles, pellets, powder, granules, fibers, membranes, substrates, tubes, bags, rings, and plates. The solid phase carrier may be packed into a column for use. The solid phase carrier is preferably made of a magnetically responsive material. If the solid phase carrier has magnetic response, it can be separated using a magnet, which is preferable as it makes it easier to separate the solid phase carrier from the liquid.

[0016] The solid phase carrier may have a metal oxide or an extracellular vesicle non-adhesive layer on at least a portion of its surface. Materials constituting the extracellular vesicle non-adhesive layer include, for example, polymers, proteins, and surfactants.

[0017] The solid phase carrier is preferably a solid phase carrier having a metal oxide on at least a part of its surface, as this further improves the adsorption rate and recovery rate of extracellular vesicles.

[0018] 1 and 2 are schematic diagrams (cross-sectional views) showing an example of a solid support. Hereinafter, the solid support will be described in more detail using as an example a particulate solid support having a metal oxide on the surface shown in FIGS.

[0019] The solid support 10 shown in FIG. 1 has a metal oxide 3 on its surface. In the solid support 10, the metal oxide 3 is present on the surface of a core portion 1. That is, the solid support 10 comprises a core portion 1 and a surface layer 4 containing the metal oxide 3. In the solid support 10, the surface layer 4 may contain micromagnetic particles 2 as shown in FIG. 1. Since the solid support 10 is a particulate solid support containing micromagnetic particles 2, it is a magnetic particle having a metal oxide 3 on its surface. The micromagnetic particles 2 may be dispersed in the surface layer 4 as shown in FIG. 1. FIG. 2 is a schematic diagram (cross-sectional view) showing another example of a solid support. As shown in FIG. 2, the micromagnetic particles 2 may be present between the metal oxide 3 and the core portion 1.

[0020] <Surface layer> The surface layer 4 includes a metal oxide 3. The surface layer 4 may further include minute magnetic particles 2, if necessary.

[0021] (metal oxides) The metal oxide 3 can be appropriately selected depending on the type of extracellular vesicles of interest, the type of sample containing extracellular vesicles to be purified, and the like.

[0022] Examples of the metal oxide 3 include oxides such as silicon dioxide (silica), glass zinc oxide, titanium oxide, nickel oxide, alumina oxide, yttria oxide, tin oxide, indium oxide, and indium tin oxide, and ceramics such as zirconia and hydroxyapatite.

[0023] Since the metal oxide 3 is more easily adsorbed to the extracellular vesicles, it is preferable that the metal oxide 3 is at least one selected from the group consisting of silicon dioxide, zinc oxide, titanium oxide, indium tin oxide, nickel oxide, yttria oxide, tin oxide, and indium oxide. In this case, the adsorption rate of the extracellular vesicles can be further improved.

[0024] From the viewpoint of facilitating detachment of extracellular vesicles at a pH near neutral, where denaturation of extracellular vesicles is unlikely to occur, it is preferable that the metal oxide 3 be at least one selected from the group consisting of silica, indium oxide, and indium tin oxide.

[0025] From the viewpoint of increasing the conductivity of the solid phase carrier and making it easier to peel off adsorbed extracellular vesicles by passing electricity through the solid phase carrier, it is preferable that the metal oxide 3 is at least one selected from the group consisting of tin oxide, indium oxide, and indium tin oxide.

[0026] From the viewpoint of facilitating the removal of phenol red contained in the culture medium and facilitating the recovery of highly pure extracellular vesicles, it is preferable that the metal oxide 3 be at least one selected from the group consisting of tin oxide, indium oxide, and indium tin oxide.

[0027] From the viewpoint of facilitating adsorption of extracellular vesicles in blood or a sample containing serum or plasma, the metal oxide 3 is preferably at least one selected from the group consisting of silica and titanium oxide.

[0028] From the viewpoint of further enhancing acid resistance and facilitating the recovery of extracellular vesicles with a high content of microRNA, the metal oxide 3 is preferably silica.

[0029] From the viewpoint of facilitating the recovery of extracellular vesicles with a high content of tetraspanins such as CD63, the metal oxide 3 is preferably at least one selected from the group consisting of indium oxide and indium tin oxide.

[0030] From the viewpoint of facilitating the recovery of extracellular vesicles with a high protein content, the metal oxide 3 is preferably titanium oxide.

[0031] The region on the surface of the solid support where the metal oxide 3 is present may be surface-treated with a compound having one or a combination of structures selected from the group consisting of anionic functional groups, cationic functional groups, hydrophobic functional groups, and polyethylene glycol groups. The compound is preferably bound to the metal oxide 3 present on the surface of the solid support by electrostatic interaction, hydrophobic interaction, hydrogen bonding, coordinate bonding, or the like. From the viewpoint of stable dispersibility, it is preferred that the various functional groups of the compound form bonds such as coordinate bonds with the metal oxide 3 present on the surface of the solid support.

[0032] By binding anionic functional groups to the metal oxide 3 present on the surface of the solid support, the surface of the solid support is more likely to be negatively charged, further suppressing the adsorption of negatively charged contaminants such as proteins, genes, and phenol red to the solid support. Furthermore, extracellular vesicles are more easily detached from the solid support, further improving the detachment rate. Suitable anionic functional groups include carboxyl, sulfo, and phosphate groups. Methods for binding anionic functional groups to the metal oxide 3 include modifying the metal oxide 3 with 3-trimethoxysilylpropyl succinic acid, 3-trimethoxysilylpropyl sulfonate, and 3-trimethoxysilylpropyl phosphonate compounds, and reacting the hydroxyl groups of the metal oxide 3 with succinic anhydride to introduce carboxyl groups.

[0033] By binding the cationic functional group to the metal oxide 3 present on the surface of the solid phase carrier, the surface of the solid phase carrier 10 is more likely to be positively charged, further suppressing the adsorption of contaminants such as positively charged proteins to the solid phase carrier. Furthermore, extracellular vesicles are more likely to be adsorbed to the solid phase carrier 10, further improving the adsorption rate. Furthermore, the recovery rate of extracellular vesicles with a high content of microRNA can be increased. Suitable examples of cationic functional groups that can be used include amino groups (unsubstituted amino groups or substituted amino groups), guanidinium groups, pyridinium groups, and imidazolium groups. Methods for bonding a cationic functional group to the metal oxide 3 include modifying the metal oxide 3 with (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-trimethoxysilylpropyl)benzyldimethylammonium chloride, (3-glycidoxypropyl)trimethoxysilane, [(2-aminoethyl)aminoethyl]triethoxysilane, and 1-imidazolylpropyltriethoxysilane, etc.

[0034] By bonding the hydrophobic functional group to the metal oxide 3 present on the surface of the solid support, the surface of the solid support becomes hydrophobic, which facilitates interaction with the hydrophobic portion of the lipids that constitute the extracellular vesicles, further improving the adsorption rate of the extracellular vesicles. Suitable hydrophobic functional groups include alkyl groups having 1 to 20 carbon atoms, such as methyl, ethyl, propyl, and butyl, as well as phenyl, alkenyl, and alkynyl groups. Methods for bonding the hydrophobic functional group to the surface of the solid support include modifying the metal oxide 3 with methyltrimethoxysilane, methyltriethoxysilane, hexyltrimethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, octyltrimethoxysilane, octyltriethoxysilane, isobutyltrimethoxysilane, hexadecyltrimethoxysilane, dimethyldiethoxysilane, and diphenyldimethoxysilane.

[0035] By attaching polyethylene glycol groups to the surface of the solid phase carrier, it is possible to further suppress the adsorption of contaminants such as proteins, genes, and phenol red to the solid phase carrier. Furthermore, when polyethylene glycol groups are attached to the surface of the solid phase carrier, it becomes easier to detach extracellular vesicles from the solid phase carrier, further improving the detachment rate. Examples of methods for modifying metal oxide 3 include methoxypolyethylene glycol, polyethylene glycol distearate, polyethylene glycol monomethyl ether, and polyethylene glycol sorbitan monooleate.

[0036] For the purpose of controlling the specific gravity of the solid phase carrier 10 and adjusting the settling rate of the solid phase carrier 10, the content of the metal oxide 3 contained in the solid phase carrier 10 is preferably 0.1 to 10 mass %, more preferably 0.5 to 5 mass %, even more preferably 1 to 5 mass %, and particularly preferably 1 to 3 mass %, based on the total mass of the solid phase carrier 10. The content of the metal oxide 3 can be measured by ICP emission spectrometry.

[0037] (micromagnetic material) The micro-magnetic bodies 2 are magnetically responsive particles smaller than the core portion. The particle size of the micro-magnetic bodies 2 may be 1 μm or less, 0.80 μm or less, 0.60 μm or less, 0.40 μm or less, 0.30 μm or less, 0.20 μm or less, or 0.15 μm or less, or may be 0.001 μm or more, 0.005 μm or more, or 0.008 μm or more. Since it is preferable that the micro-magnetic bodies 2 have superparamagnetism, the particle size of the micro-magnetic bodies 2 is preferably 0.001 to 1 μm, more preferably 0.001 to 0.5 μm, even more preferably 0.001 to 0.1 μm, and particularly preferably 0.001 to 0.05 μm. The micro-magnetic bodies 2 are preferably nano-sized magnetic bodies (nano-magnetic bodies).

[0038] The particle size of the minute magnetic particles 2 can be calculated by measuring the maximum particle diameter of 10 or more particles in an image observed with a transmission electron microscope and calculating the average value.

[0039] The minute magnetic bodies 2 may be made of, for example, iron oxide such as magnetite.

[0040] The surface of the magnetic microparticles 2 may contain an inorganic surface modifier such as lipids and a silane coupling agent. Examples of silane coupling agents include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, bis(triethoxysilylpropyl)tetrasulfide, and 3-isocyanatepropyltriethoxysilane. The surface of the minute magnetic particles 2 may contain a cationic dispersant or an anionic dispersant.

[0041] Commercially available products can be used as the micro magnetic bodies 2. An example of a commercially available micro magnetic body 2 is EMG607 (trade name, manufactured by Ferrotec Corporation).

[0042] The micro magnetic particles 2 may be in a state of being dispersed in the surface layer 4, or may be in a state of being located between the core portion 1 and the layer consisting of the metal oxide 3, covering the core portion and being covered by the metal oxide 3. When the micro magnetic particles 2 are in a state of being dispersed in the surface layer 4, or when the surface of the micro magnetic particles 2 is in a state of being covered by the metal oxide 3, the acid resistance of the solid phase carrier 10 can be improved.

[0043] Since the magnetic response is further improved, the content of the micro-magnetic particles 2 per 1 g of the solid phase carrier is preferably 0.1 g / g or more, more preferably 0.2 g / g or more, even more preferably 0.5 g / g or more, and particularly preferably 0.7 g / g or more. Since the dispersibility of the solid phase carrier 10 in aqueous solvents is further improved, the content of the micro-magnetic particles 2 per 1 g of the solid phase carrier is preferably 0.9 g / g or less, more preferably 0.8 g / g or less, even more preferably 0.7 g / g or less, and particularly preferably 0.6 g / g or less.

[0044] From the viewpoint of controlling the specific gravity of the solid phase carrier 10 and facilitating adjustment of the sedimentation rate of the solid phase carrier 10, the weight proportion of the micromagnetic particles 2 contained in the solid phase carrier 10 is preferably 5 to 70 mass %, more preferably 10 to 60 mass %, even more preferably 15 to 50 mass %, and particularly preferably 20 to 40 mass %, based on the total mass of the solid phase carrier 10. The content of the micromagnetic particles 2 can be measured by ICP (Inductively Coupled Plasma) emission spectrometry.

[0045] When a metal oxide 3 is coated along the uneven structure formed by the magnetic particles 2 located on the surface of the core 1, a surface layer 4 having an uneven structure can be formed. When the surface layer 4 has an uneven structure on its surface, the specific surface area of ​​the solid phase carrier 10 increases, and the amount of extracellular vesicles that can be carried can be increased.

[0046] (Surface layer thickness) The thickness of the surface layer 4 may be, for example, 0.001 μm or more and 0.1 μm or less. From the viewpoint of further improving acid resistance, the thickness of the surface layer 4 is preferably 0.001 μm or more, more preferably 0.005 μm or more, even more preferably 0.01 μm or more, and particularly preferably 0.05 μm or more. From the viewpoint of further increasing the specific surface area of ​​the solid phase carrier 10, the thickness of the surface layer 4 is preferably 0.1 μm or less, more preferably 0.05 μm or less, even more preferably 0.02 μm or less, and particularly preferably 0.01 μm or less. The thickness of the surface layer 4 can be calculated by measuring the thickness of the surface layer 4 at 10 or more points on the solid phase carrier 10 in an image of the cross section of the solid phase carrier 10 observed with a transmission electron microscope and calculating the average value.

[0047] <Core> The shape of the core part 1 is not limited to particles, and can be selected as appropriate from pellets, powders, granules, fibers, films, substrates, tubes, bags, rings, plates, etc. Since this is suitable for coating the surface of the core part 1 with many minute magnetic bodies, it is preferable that the core part 1 has irregularities with a large specific surface area, such as a porous shape or one with protrusions.

[0048] Examples of the shape of the particulate core portion 1 include spherical shapes close to a perfect sphere, non-spherical shapes such as ellipsoids, cubes, cylinders, and polygonal prisms, and particles with large specific surface areas and irregularities such as porous shapes and those with protrusions. Since magnetic particles with good magnetic response are easily obtained, the particle size of the core portion 1 is preferably 0.1 μm or more, more preferably 0.5 μm or more, even more preferably 1.0 μm or more or 1.5 μm or more, and particularly preferably 2.0 μm or more. Since magnetic particles with good redispersibility in aqueous solvents are easily obtained, the particle size of the core portion 1 is preferably 100 μm or less, more preferably 80 μm or less, 60 μm or less, 40 μm or less, 20 μm or less, or 10 μm or less, even more preferably 5 μm or less, and particularly preferably 3 μm or less. The particle size of the core portion 1 may be 0.1 μm or more and 100 μm or less, 0.5 μm or more and 10 μm or less, 1.0 μm or more and 5.0 μm or less, or 2.0 μm or more and 3.0 μm or less. The particle size of the core portion 1 can be calculated by determining the mode diameter from the particle size distribution measured by dynamic light scattering.

[0049] The core 1 may be made of a polymer or a metal oxide. When the core 1 is made of a polymer, the specific gravity of the solid carrier 10 can be reduced compared to when the main component of the core 1 is a component with a high specific gravity (a magnetic material or a metal oxide), and the dispersibility of the solid carrier 10 in a solvent is improved. The improved dispersibility makes it easier to obtain particulate solid carriers 10 that can be uniformly dispersed with little stirring force. A particulate solid carrier that can be uniformly dispersed can improve the reproducibility of extracellular vesicle purification. The metal oxide that constitutes the core 1 may be any of the materials exemplified as the metal oxide in the surface layer 4.

[0050] The polymer may contain, as a monomer unit, for example, at least one selected from the group consisting of a styrene-based monomer unit, a (meth)acrylate-based monomer unit, and a vinyl ester-based monomer.

[0051] Examples of styrene-based monomers include styrene, α-methylstyrene, vinyltoluene, p-methylstyrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, 4-tert-butylstyrene, 3,4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4-chloro-3-methylstyrene, divinylbenzene, and sodium p-styrenesulfonate.

[0052] Examples of (meth)acrylate monomers include (meth)acrylate ((meth)acrylic acid); alkyl (meth)acrylates such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, and 2-ethylhexyl (meth)acrylate; (cyclo)alkyl (meth)acrylates such as cyclohexyl (meth)acrylate; 2-methoxyethyl (meth)acrylate, p-methoxycyclohexyl (meth)acrylate; Alkoxy(cyclo)alkyl(meth)acrylates such as cyclo(meth)acrylate; poly(meth)acrylates such as trimethylolpropane tri(meth)acrylate; cyanoacrylates such as 2-cyanoethyl(meth)acrylate, 2-cyanopropyl(meth)acrylate, 3-cyanopropyl(meth)acrylate; hydroxymethyl(meth)acrylate, 2-hydroxyethyl(meth)acrylate, 6-hydroxyhexyl(meth)acrylate, 4-hydroxypropyl ... Examples of suitable hydroxy (meth)acrylates include substituted hydroxy (meth)acrylates such as cyclohexyl (meth)acrylate, neopentyl glycol mono (meth)acrylate, 3-chloro-2-hydroxypropyl (meth)acrylate, and 3-amino-2-hydroxypropyl (meth)acrylate; glycidyl group-containing acrylates such as glycidyl (meth)acrylate, methyl glycidyl methyl acrylate, and epoxidized cyclohexyl (meth)acrylate; and polyfunctional (meth)acrylates such as trimethylolpropane ethoxy triacrylate, pentaerythritol ethoxy tetraacrylate, trimethylolpropane propoxy triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, dipentaerythritol hexaacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, tricyclodecane dimethanol diacrylate, and ethoxylated phenyl acrylate. The (meth)acrylate may be crosslinked.

[0053] Examples of the vinyl ester monomer include vinyl acetate, vinyl propionate, and vinyl versatate.

[0054] The core portion 1 may contain minute magnetic substances as needed. As the minute magnetic substances that can be contained in the surface layer 4, those exemplified above can be used.

[0055] A paramagnetic thin film coating layer may be provided between the core portion 1 and the surface layer 4. By providing a paramagnetic coating layer, the magnetic responsiveness of the solid phase carrier 10 can be improved.

[0056] When a paramagnetic coating layer is provided, the thickness of the paramagnetic coating layer is preferably 0.001 to 1 μm, more preferably 0.001 to 0.1 μm, even more preferably 0.001 to 0.01 μm, and particularly preferably 0.001 to 0.005 μm, since this is suitable for reducing residual magnetization in the core part 1 and suppressing aggregation of the core parts 1. The thickness of the paramagnetic coating layer can be calculated by measuring the thickness of the layer at 10 or more points of the core part 1 in an image of the cross section of the core part 1 observed with a transmission electron microscope and calculating the average value.

[0057] As a pretreatment for providing a paramagnetic coating layer, a substance having a positive or negative charge may be coated on the surface of the core part 1. There are no particular limitations on the method for coating the surface of the core part 1 with a substance having a positive or negative charge, but examples include a method of forming a polymer layer on the surface of the core part 1 using a monomer having a charge in its side chain, and a method of coating the surface of the core part 1 with a charged polymer by a layer-by-layer method.

[0058] (Specific surface area of ​​solid support) The specific surface area of ​​the solid support 10 is 2 m 2 / g or more. 2 / g or more, the amount of extracellular vesicles that can be bound to the surface of the solid phase carrier 10 per unit weight can be increased. Therefore, the large specific surface area of ​​the solid phase carrier 10 makes it possible to adsorb extracellular vesicles with a small amount of solid phase carrier 10. The specific surface area of ​​the solid phase carrier 10 is 5m 2 / g or more is more preferable, and 10m 2 / g or more is more preferable, 2 The specific surface area of ​​the solid support is preferably 100 m / g or more, as this is suitable for increasing the mechanical strength of the solid support. 2 / g or less, 90m 2 / g or less, 80m 2 / g or less, 70m 2 / g or less, or 60m 2 / g or less, and 2 / g or less is more preferable, and 40m 2 / g or less is more preferable, 2 The specific surface area can be calculated from the amount of adsorption of an inert gas by the BET method of JIS Z8830:2013.

[0059] <Method of manufacturing a solid support having a metal oxide on its surface> The solid support 10 having a metal oxide on the surface thereof can be obtained, for example, by a method including a step of coating the core portion 1 with a metal oxide 3 (coating step).

[0060] When the solid phase carrier 10 contains the micro magnetic particles 2, the manufacturing method of the solid phase carrier 10 may further include a step of incorporating the micro magnetic particles 2 into the solid phase carrier 10 (a magnetization step). Examples of methods for incorporating the micro magnetic particles 2 into the solid phase carrier 10 include a method of adsorbing the micro magnetic particles 2 onto the surface of the core part 1 before the coating step, and a method of adding the micro magnetic particles when coating the core part with a metal oxide in the coating step.

[0061] (Magnetization process) The magnetization step may be a step of adsorbing (physical adsorbing) the micro-magnetic body to the surface of the core part by contacting the core part 1 with the micro-magnetic body 2. The method of contacting the core part 1 with the micro-magnetic body 2 is not particularly limited, but can be carried out by stirring in a liquid phase or a gas phase.

[0062] In the magnetization process, the amount of micro-magnetic material 2 used may be, for example, 0.5 g or more, 0.6 g or more, 0.7 g or more, 0.8 g or more, 0.9 g or more, 1.0 g or more, or 1.1 g or more per 1 g of core portion, and may be 2.0 g or less, 1.9 g or less, 1.8 g or less, 1.7 g or less, 1.6 g or less, or 1.5 g or less.

[0063] Examples of methods for adsorbing the micromagnetic substance 2 onto the surface of the core part 1 include a method in which the core part 1 is brought into contact with a micromagnetic substance 2 having a surface charge opposite to that of the core part 1, thereby spontaneously adsorbing the micromagnetic substance 2 onto the surface of the core part 1 through electrostatic interaction, a method in which the micromagnetic substance 2 is bonded to the surface of the core part 1 through a chemical reaction between a functional group on the surface of the core part 1 and the micromagnetic substance 2, a method in which the core part 1 and the micromagnetic substance 2 are combined by applying mechanical energy, a method in which a hydrophobic core part 1 is brought into contact with a hydrophobic micromagnetic substance 2, and a method in which a paramagnetic core part 1 is brought into contact with the micromagnetic substance 2. The method for adsorbing the micromagnetic substance 2 onto the surface of the core part 1 may be a combination of several methods. For example, the micromagnetic substance 2 may be spontaneously adsorbed onto the surface of the core part 1 through electrostatic interaction, dried, and then further mechanical energy may be applied to firmly adsorb the micromagnetic substance 2 onto the core part 1.

[0064] As a method for spontaneously adsorbing the micro-magnetic particles 2 onto the surface of the core part 1 through electrostatic interaction, it is preferable to mix the core part 1 and the micro-magnetic particles 2 in an aqueous solution, as this is suitable for increasing the amount of the micro-magnetic particles 2 adsorbed to the core part 1. When the core part 1 and the micro-magnetic particles 2 are mixed in an aqueous solution, it is preferable that the aqueous solution contains an electrolyte such as sodium chloride. When the aqueous solution contains an electrolyte, the electrolyte concentration in the aqueous solution is preferably a concentration at which the micro-magnetic particles 2 do not aggregate with each other, and for example, a concentration of about 0.01 to 0.5 M can be suitably used.

[0065] The reaction conditions (reaction temperature and reaction time) when adsorbing the micro magnetic particles 2 onto the surface of the core part 1 by electrostatic interaction can be set appropriately depending on the type of material used, etc. The reaction temperature when adsorbing the micro magnetic particles 2 onto the surface of the core part 1 may be, for example, 1 to 50°C, 5 to 40°C, 10 to 30°C, or 15 to 25°C. The reaction time when adsorbing the micro magnetic particles 2 onto the surface of the core part 1 may be, for example, 1.0 hour or more, 1.5 hours or more, 2.0 hours or more, 3.0 hours or more, or 4.0 hours or more, or may be 5.0 hours or less, 4.0 hours or less, or 3.0 hours or less.

[0066] Preferred methods for compounding the core 1 and the magnetic particles 2 by applying mechanical energy include dry processing using a device that uses swirling force in a high-speed airflow to compound the particles, a device that mixes and stirs the particles using a rotation-revolution mixer or ball mill, or a device that coats the particles using a spray dryer. Examples of devices for compounding the particles by applying mechanical energy include the Hybridization System NHS Series (manufactured by Nara Machinery Works, Ltd.), Nobilta NOB (manufactured by Hosokawa Micron Corporation), the High-Speed ​​Agitation Powder Spheroidizer NSM Series (manufactured by Seishin Enterprise Co., Ltd.), and the Mini Spray Dryer B-290 (manufactured by Shibata Scientific Co., Ltd.).

[0067] When compounding is performed by the rotational force in a high-speed airflow, the rotation speed is set to 8000 min -1 It is preferable that the speed is 9000 min or more. -1 More preferably, it is 10,000 min or more. -1 More preferably, it is 11,000 min or more. -1 The rotation speed is preferably 15,000 min or more, as this is suitable for forming the particles into a spherical shape. -1 It is preferable that the temperature is less than 14,000 min -1 It is more preferable that it is less than 13,000 min -1 More preferably, it is 12,000 min or less. -1The following is particularly preferred. Since this is suitable for forming the particles into a spherical shape, the treatment time is preferably 1 to 60 minutes, more preferably 1 to 20 minutes, even more preferably 1 to 10 minutes, and particularly preferably 3 to 10 minutes. Since this is suitable for suppressing fusion of the particles, the treatment temperature is preferably 25 to 80°C, more preferably 25 to 60°C, even more preferably 25 to 50°C, and particularly preferably 25 to 40°C.

[0068] It is preferable to electrostatically adsorb the minute magnetic particles 2 to the core portion 1 in an aqueous solution, as this is suitable for uniformly adsorbing the minute magnetic particles 2 to the core portion.

[0069] In order to more firmly fix the adsorbed micromagnetic particles 2 to the surface of the core portion 1, it is preferable to dry the material after the composite in the aqueous solution. Drying conditions can be appropriately set depending on the type of material used, the composite conditions, etc. The drying temperature may be, for example, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and may be 80°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, or 55°C or lower. The drying time may be, for example, 0.5 hours or higher, 1 hour or higher, 3 hours or higher, 5 hours or higher, or 10 hours or higher, and may be 20 hours or lower, 15 hours or lower, 10 hours or lower, or 5 hours or lower.

[0070] The core parts 1 with the micro-magnetic particles 2 adsorbed thereto may be dispersed in a high-speed air stream after drying. By dispersing the core parts 1 with the micro-magnetic particles 2 adsorbed thereto in a high-speed air stream, the aggregates of the core parts 1 can be dissociated by the impact force. While dispersing the core parts 1 with the micro-magnetic particles 2 adsorbed thereto in a high-speed air stream, the core parts 1 can be surface-modified in a dry manner by the impact force between the core parts 1, thereby making the shape of the core parts 1 spherical. The high-speed air stream treatment can be performed using the above-mentioned device that performs compounding by swirling force in a high-speed air stream.

[0071] (Coating process) The coating step is a step of coating the core part 1 with a metal oxide 3. The method of coating the core part 1 with the metal oxide 3 is not particularly limited, but examples include a method of dispersing the core part 1, on which the fine magnetic particles 2 are adsorbed, in a solution containing a reagent and a reaction catalyst that are raw materials for the metal oxide 3, and allowing the reaction to proceed to form the metal oxide 3 on the surface of the core part 1, a method of dispersing the powder of the core part 1, on which the fine magnetic particles 2 are adsorbed, and the metal oxide 3 in a high-velocity airflow, to form the metal oxide 3 on the surface of the core part 1, and a method of coating the surface of the core part 1 with the metal oxide or its raw material.

[0072] A suitable method for coating the core 1 with the metal oxide 3 is a sol-gel method in which the core 1 having the magnetic particles 2 adsorbed thereon is dispersed in a solvent, and then a precursor of the metal oxide 3 and a reaction catalyst are added. Examples of the precursor of the metal oxide 3 include metal alkoxides. Examples of precursors of the metal oxide 3 include tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate for silica; zinc methoxide, zinc ethoxide, zinc propoxide, and zinc butoxide for zinc oxide; tetramethyl orthotitanate, tetraethyl orthotitanate, tetrapropyl orthotitanate, and tetrabutyl orthotitanate for titanium oxide; yttrium methoxide, yttrium ethoxide, yttrium propoxide, and yttrium butoxide for yttria oxide; tin methoxide, tin ethoxide, tin propoxide, and tin butoxide for tin oxide; and indium methoxide, indium ethoxide, indium propoxide, and indium butoxide for indium oxide. In the case of indium tin oxide, a mixture of a tin oxide precursor and an indium oxide precursor can be used. Water or aqueous ammonia can be used as a reaction catalyst. The amount of the precursor of metal oxide 3 added during the sol-gel reaction is preferably 0.05 g or more, more preferably 0.1 g or more, more preferably 0.2 g or more, and most preferably 0.5 g or more per 1 g of core portion, because this is suitable for forming a sufficient amount of metal oxide 3 on the surface of core portion 1. The amount of the precursor of metal oxide 3 added during the sol-gel reaction is preferably 5 g or less, more preferably 2 g or less, particularly preferably 1 g or less, and most preferably 0.5 g or less per 1 g of core portion, because this is suitable for suppressing aggregation of core portion 1 during the reaction. The amount of the reaction catalyst added during the reaction is preferably 20 mL or more, more preferably 30 mL or more, particularly preferably 40 mL or more, and most preferably 50 mL or more per 1 L of solvent, because this is suitable for forming a sufficient amount of metal oxide on the surface of core portion 1.Since this is suitable for suppressing aggregation of the core part 1 during the reaction, the amount of reaction catalyst added during the reaction is preferably 100 mL or less per 1 L of solvent, more preferably 80 mL or less, particularly preferably 70 mL or less, and most preferably 60 mL or less.

[0073] As a method for coating the core portion 1 with the metal oxide 3, a method can also be used in which particles are dispersed in a solution containing metal ions, and the metal oxide 3 is precipitated to coat the surface of the core portion 1 with the metal oxide 3. To precipitate the metal oxide 3, a metal salt and a precipitating agent for precipitation are used. The metal salt can be a salt of a metal ion and its counterion. Examples of counterions to metal ions include nitrate ions, acetate ions, and sulfate ions. Examples of metal salts that can be used include zinc nitrate, zinc sulfate, and zinc acetate for zinc oxide; titanium nitrate, titanium sulfate, and titanium acetate for titanium oxide; yttrium nitrate, yttrium sulfate, and yttrium acetate for yttria oxide; tin nitrate, tin sulfate, and tin acetate for tin oxide; and indium nitrate, indium sulfate, and indium acetate for indium oxide. In the case of indium tin oxide, a mixture of a metal salt that provides tin oxide and a metal salt that provides indium oxide can be used. Examples of precipitating agents that can be used include alkali metal hydroxides, aqueous ammonia, and alkalis such as hexamethylenetetramine.

[0074] As a reaction solvent for forming the metal oxide 3, ethanol, 2-propanol, or butanol is preferred, as these are suitable for uniformly coating the metal oxide 3 while suppressing aggregation of the core portion 1, with ethanol or 2-propanol being more preferred, and 2-propanol being particularly preferred.

[0075] As a method for coating the core part 1 with the metal oxide 3, powder sputtering can also be used, in which the dried core part 1 is sputtered with the metal oxide 3 while being stirred in a drum or the like. In this case, the coating time is preferably 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.

[0076] <Solid support with polymer on the surface> The solid support may have a polymer on at least a portion of its surface. The polymer may be a polymer containing at least one monomer unit selected from the group consisting of styrene-based monomer units and (meth)acrylate-based monomer units. Specific examples of the styrene-based monomer units and (meth)acrylic acid monomer units are as described above. Furthermore, the polymer may contain a monomer unit having a polyethylene glycol group or a polyoxyethylene group, or a surfactant, as necessary.

[0077] Examples of the monomer having a polyethylene glycol group or a polyoxyethylene group include polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, polyethylene glycol methyl ether acrylate, and polyethylene glycol methyl ether methacrylate.

[0078] Examples of surfactants having a polyethylene glycol group or a polyoxyethylene group include polyoxyethylene alkyl ethers, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene myristyl ether, polyoxyethylene alkylene alkyl ethers, polyoxyethylene phenyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene polyoxypropylene, polyoxyethylene polyoxypropylene alkyl ethers, polyethylene glycol alkyl esters, polyoxyalkylene glycol rosinate esters, polyoxyethylene alkylamines, and polyethylene glycol, with polyoxyethylene alkyl ethers having a long alkyl chain of 1 to 20 carbon atoms being preferred.

[0079] The molecular weight of the polyethylene glycol or oxyethylene is preferably 500 or more, more preferably 1000 or more, more preferably 1500 or more, and particularly preferably 2000 or more. Furthermore, the molecular weight of the polyoxyethylene is preferably 20000 or less, more preferably 10000 or less, particularly preferably 5000 or less, and most preferably 3000 or less.

[0080] The polymer may be a polymer containing (meth)acrylic acid monomer units and (meth)acrylic acid ester monomer units, or may be a polymer containing styrene-based monomer units and (meth)acrylic acid monomer units.

[0081] The content of the styrene-based monomer units may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, 70 parts by mass or more, or 90 parts by mass or more, and may be 95 parts by mass or less, 75 parts by mass or less, 55 parts by mass or less, 35 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0082] The content of the (meth)acrylate monomer units may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, 70 parts by mass or more, or 90 parts by mass or more, and may be 95 parts by mass or less, 75 parts by mass or less, 55 parts by mass or less, 35 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0083] The content of the (meth)acrylic acid monomer unit may be 1 part by mass or more, 5 parts by mass or more, 10 parts by mass or more, 20 parts by mass or more, or 40 parts by mass or more, and may be 50 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, or 10 parts by mass or less, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0084] The content of the (meth)acrylic acid ester monomer units may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, 70 parts by mass or more, or 90 parts by mass or more, and may be 95 parts by mass or less, 75 parts by mass or less, 55 parts by mass or less, 35 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0085] The amount of the monomer or surfactant having a polyethylene glycol structure or a polyoxyethylene structure may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, 70 parts by mass or more, or 90 parts by mass or more, relative to 100 parts by mass of the total amount of monomer units constituting the polymer, and may be 95 parts by mass or less, 75 parts by mass or less, 55 parts by mass or less, 35 parts by mass or less, or 15 parts by mass or less.

[0086] A solid support having a polymer on its surface can be obtained by forming a polymer on the surface of the solid support by a polymerization reaction of a monomer in a reaction solution containing the solid support, a monomer, and an initiator (polymerization initiator). The reaction solution may contain additives such as surfactants as needed. Examples of surfactants include nonionic surfactants such as polyoxyethylene lauryl ether. The solid support used to form a polymer on its surface may be treated with a silane coupling agent as needed. Specific examples of silane coupling agents are as described above.

[0087] [(B) component] Component (B) is a component containing two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions. Component (B) is a component containing carbonate ions (CO3 2- ), phosphate ions (PO4 3- ) and calcium ions (Ca 2+ In this specification, the carbonate ion that can be contained as component (B) includes carbonic acid and ions generated by dissociation of carbonic acid. In component (B), the carbonate ion is H2CO3, HCO3 - and CO3 2- In this specification, the phosphate ions that can be contained as component (B) include phosphoric acid and ions generated by dissociation of phosphoric acid. In component (B), the phosphate ions are H3PO4, H2PO4 - , HPO4 2- , and PO4 3- It may exist in either state.

[0088] Component (B) may contain an independent compound containing one type of ion selected from carbonate ions, phosphate ions, and calcium ions, or may contain a compound containing two or more types of ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions (e.g., calcium carbonate salts, calcium phosphate salts, and complex inorganic salts thereof).

[0089] By using the above component (B) in the purification of extracellular vesicles, the adsorption rate of extracellular vesicles to a solid phase carrier can be increased. Therefore, component (B) can also be called an adsorption promoter.

[0090] By adjusting the amounts of two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions used, it is possible to control the adsorption of extracellular vesicles to solid phase carriers and the desorption of extracellular vesicles from the solid phase carriers to which the extracellular vesicles are adsorbed. When extracellular vesicles are adsorbed to solid phase carriers in the presence of two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions, washing the solid phase carriers to which the extracellular vesicles are adsorbed with an aqueous solution containing the two or more ions can prevent the extracellular vesicles from being desorbed from the solid phase carrier and being lost during washing. The ease of desorption of extracellular vesicles from solid phase carriers is affected by the adsorption conditions. However, when two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions are used, the extracellular vesicles can be easily desorbed and recovered from the solid phase carriers by contacting the solid phase carriers with a liquid that does not contain the two or more ions.

[0091] Since the adsorption rate and recovery rate of extracellular vesicles are further improved, component (B) may be a component containing calcium ions and carbonate ions and / or phosphate ions, a component containing at least calcium ions and phosphate ions, or a component containing all of carbonate ions, phosphate ions, and calcium ions.

[0092] Examples of component (B) include a component containing a carbonate, a phosphate, and a calcium salt, a component containing a carbonate and calcium phosphate, and a component containing calcium carbonate and a phosphate. Component (B) is preferably a component containing a carbonate, a phosphate, and a calcium salt, as this further improves the adsorption rate and recovery rate of extracellular vesicles.

[0093] Carbonates are CO3 2- or HCO3 - and its counterion. 2- or HCO3 -Counter ions include alkali metal ions (e.g., lithium ion, sodium ion, and potassium ion), alkaline earth metal ions (e.g., magnesium ion, calcium ion, strontium ion, and barium ion), and ammonium ion. Carbonates include, for example, sodium bicarbonate, calcium carbonate, magnesium carbonate, potassium carbonate, lithium carbonate, ammonium carbonate, strontium carbonate, and barium carbonate. The carbonate is preferably sodium bicarbonate, as this is suitable for increasing the adsorption rate of extracellular vesicles, suppressing loss during washing, and increasing the recovery rate of extracellular vesicles from the solid phase carrier.

[0094] Phosphate is PO4 3- , HPO4 2- or H2PO 4- and its counterion. 3- , HPO4 2- or H2PO 4- Counter ions of the above include, for example, alkali metal ions and alkaline earth metal ions. Examples of phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, calcium hydrogen phosphate, potassium hydrogen phosphate, magnesium hydrogen phosphate, ammonium hydrogen phosphate, strontium hydrogen phosphate, and barium hydrogen phosphate. Since the phosphate is suitable for increasing the adsorption rate of extracellular vesicles, suppressing loss during washing, and increasing the recovery rate of extracellular vesicles from the solid phase carrier, it is preferable that the phosphate is at least one selected from the group consisting of dihydrogen phosphate and hydrogen phosphate, or at least one selected from the group consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate, and sodium dihydrogen phosphate is more preferable.

[0095] Calcium salts are Ca 2+ It is a compound consisting of Ca and its counter ion. 2+Examples of the counter ion include inorganic acid ions (e.g., halide ions, nitrate ions, sulfate ions) and organic acid ions (e.g., acetate ions). Examples of calcium salts include calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium sulfate, and calcium acetate. The calcium salt is preferably calcium chloride, as it is suitable for increasing the adsorption rate of extracellular vesicles, suppressing loss during washing, and increasing the recovery rate of extracellular vesicles from the solid phase carrier.

[0096] The content of carbonate per 1 mol of phosphate is preferably 0.1 mol or more, 0.5 mol or more, 1 mol or more, 2 mol or more, 5 mol or more, 10 mol or more, 15 mol or more, 20 mol or more, 25 mol or more, 30 mol or more, 35 mol or more, 40 mol or more, or 45 mol or more, because this can further promote the adsorption of extracellular vesicles to the solid phase carrier and further improve the recovery rate of extracellular vesicles, and may be, for example, 50 mol or more or 100 mol or more.

[0097] The content of carbonate per 1 mol of phosphate is preferably 200 mol or less, 150 mol or less, 120 mol or less, 80 mol or less, 60 mol or less, 55 mol or less, or 50 mol or less, because this can further promote the adsorption of extracellular vesicles to the solid phase carrier and further improve the recovery rate of extracellular vesicles, and may be, for example, 45 mol or less, 40 mol or less, 30 mol or less, 15 mol or less, 8 mol or less, or 4 mol or less.

[0098] The content (mol) of carbonate ions per 1 mol of phosphate ions may be in the same numerical range as the "content of carbonate per 1 mol of phosphate" described above.

[0099] The content of carbonate per 1 mol of calcium salt is preferably 0.05 mol or more, 0.15 mol or more, 0.3 mol or more, 1 mol or more, 3 mol or more, 6 mol or more, 8 mol or more, 10 mol or more, 12 mol or more, 15 mol or more, 18 mol or more, 20 mol or more, or 22 mol or more, because this can further promote the adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles, and may be, for example, 25 mol or more or 50 mol or more.

[0100] The content of carbonate per 1 mol of calcium salt is preferably 150 mol or less, 120 mol or less, 80 mol or less, 70 mol or less, 60 mol or less, 55 mol or less, 50 mol or less, 45 mol or less, 40 mol or less, 35 mol or less, 30 mol or less, or 25 mol or less, because this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles, and may be, for example, 20 mol or less, 10 mol or less, 5 mol or less, 2 mol or less, or 1 mol or less.

[0101] The content (mol) of carbonate ions per 1 mol of calcium ions may be in the same numerical range as the "content of carbonate salt per 1 mol of calcium salt" described above.

[0102] The content of calcium salt per 1 mol of phosphate is preferably 0.1 mol or more, 0.3 mol or more, 0.5 mol or more, 0.8 mol or more, 1 mol or more, 1.2 mol or more, 1.4 mol or more, 1.5 mol or more, 1.6 mol or more, or 1.8 mol or more, as this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles, and may be, for example, 2 mol or more, 3 mol or more, or 5 mol or more.

[0103] The content of calcium salt per 1 mol of phosphate is preferably 20 mol or less, 18 mol or less, 16 mol or less, 14 mol or less, 12 mol or less, 10 mol or less, 8 mol or less, 6 mol or less, 4 mol or less, 3 mol or less, 2.5 mol or less, or 2 mol or less, since this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles, and may be, for example, 1 mol or less, 0.7 mol or less, 0.4 mol or less, or 0.2 mol or less.

[0104] The content (mol) of calcium ions per mol of phosphate ions may be in the same numerical range as the "content of calcium salt per mol of phosphate" described above.

[0105] The total content of component (B) (and the combined content of carbonate, phosphate, and calcium salt) per gram of solid phase carrier may be 0.01 mmol or more, 0.05 mmol or more, 0.1 mmol or more, 0.5 mmol or more, 1 mmol or more, 5 mmol or more, 10 mmol or more, 100 mmol or more, or 1000 mmol or more, preferably 2 mmol or more, 3 mmol or more, or 4 mmol or more, or 2000 mmol or less, 200 mmol or less, 20 mmol or less, 15 mmol or less, 5 mmol or less, 2 mmol or less, 0.8 mmol or less, 0.5 mmol or less, 0.2 mmol or less, 0.08 mmol or less, or 0.05 mmol or less, preferably 8 mmol or less, 7 mmol or less, or 6 mmol or less.

[0106] The total number of moles of carbonate ions, phosphate ions, and calcium ions per gram of solid phase carrier may be in the same numerical range as the "total content of component (B) (and the total content of carbonate, phosphate, and calcium salt)" described above.

[0107] Component (B) may be in a solid state (powder, etc.) or in a solution state. Component (B) in a solution state can be prepared by dissolving at least two or more species selected from the group consisting of carbonates, phosphates, and calcium salts in a solvent such as water. Component (B) in a solution state can be diluted as necessary before use in purifying extracellular vesicles.

[0108] In the extracellular vesicle purification kit, component (B) may be mixed with component (A), or may not be mixed with component (A).

[0109] <(C) component> The extracellular vesicle purification kit may further contain (C) a chelating agent (hereinafter also referred to as "component (C)"). Use of a chelating agent in the purification of extracellular vesicles promotes the desorption of extracellular vesicles from the solid phase carrier to which the extracellular vesicles are adsorbed. Therefore, component (C) can also be referred to as a desorption promoter.

[0110] Chelating agents include EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), NTA (naphthyltriamineacetic acid), EGTA (ethylene glycol bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid), BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), citric acid, desferrioxamine, penicillamine, and salts thereof.

[0111] The total content of the chelating agent is preferably 0.001 μmol or more, 0.005 μmol or more, 0.01 μmol or more, 0.02 μmol or more, 0.05 μmol or more, 0.1 μmol or more, 0.5 μmol or more, or 1 μmol or more per 1 g of solid phase carrier. In order to further promote the detachment of extracellular vesicles and suppress the denaturation of extracellular vesicles, the total content of the chelating agent is preferably 10 μmol or less, 5 μmol or less, 3 μmol or less, 1 μmol or less, 0.8 μmol or less, 0.4 μmol or less, 0.2 μmol or less, 0.15 μmol or less, 0.08 μmol or less, 0.04 μmol or less, 0.015 μmol or less, 0.008 μmol or less, or 0.004 μmol or less per 1 g of solid phase carrier.

[0112] Component (C) may be in a solid state (powder, etc.) or in a solution state. Component (C) in a solution state can be prepared by dissolving the chelating agent in a solvent such as water. Component (C) in a solution state can be diluted as necessary before use in purifying extracellular vesicles.

[0113] In the extracellular vesicle purification kit, component (C) may be mixed with component (A) and / or component (B), or may not be mixed with component (A) and / or component (B).

[0114] <Other ingredients> The extracellular vesicle purification kit may further contain other components in addition to components (A) to (C). Examples of such other components include a pH adjuster (excluding components corresponding to component (B)) and a protein remover. The protein remover is used as a pretreatment to remove proteins from a liquid containing extracellular vesicles. The type of protein remover is not particularly limited, and suitable examples include ammonium sulfate, trichloroacetic acid, acetone, chloroform, methanol, phenol, and mixtures thereof.

[0115] [Extracellular vesicle purification method] The method for purifying extracellular vesicles according to this embodiment is a method for purifying extracellular vesicles using the above-described extracellular vesicle purification kit. The purification method according to this embodiment includes a step of mixing a liquid containing extracellular vesicles with component (A) and component (B), an adsorption step of adsorbing extracellular vesicles on the surface of component (A), a separation step of separating the (A) component adsorbed with extracellular vesicles from the liquid, a washing step of washing the (A) component adsorbed with extracellular vesicles, and a desorption step of desorbing extracellular vesicles from the (A) component adsorbed with extracellular vesicles.

[0116] The method for purifying extracellular vesicles may further include a pH adjustment step of adjusting the pH of the liquid containing extracellular vesicles to an acidic pH (making it an acidic liquid) before the adsorption step.

[0117] Hereinafter, the method for purifying extracellular vesicles will be described in more detail by taking as an example a method including the pH adjustment step, adsorption step, separation step, washing step, and desorption step in this order.

[0118] <pH adjustment step> In the pH adjustment step, the pH of the liquid containing extracellular vesicles is adjusted to make it an acidic liquid. In the pH adjustment step, by setting it to an acidic pH (hydrogen ion index), the solid phase carrier and proteins are likely to carry a positive charge, while extracellular vesicles are negatively charged even in an acidic environment, making it easy to selectively adsorb extracellular vesicles on the surface of the solid phase carrier.

[0119] Examples of the liquid containing extracellular vesicles include, for example, a medium in which cells are cultured (culture supernatant), blood, serum, plasma, urine, sweat, saliva, breast milk, and the like.

[0120] The method for adjusting the pH is not particularly limited, and examples include a method of adding an acid such as phosphoric acid, sodium dihydrogen phosphate, disodium phosphate, tris(tris(hydroxymethyl)aminomethane), hydrochloric acid, acetic acid, sodium acetate, 2-morpholinoethanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, trishydroxymethylaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3-tris(hydroxymethyl)methylaminopropanesulfonic acid, boric acid, sodium borate, 2-(N-morpholino)ethanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), acetamidoimidole, N-(2-acetamido)-2-aminoethanesulfonic acid, and 2-(N-cyclohexylamino)ethanesulfonic acid. To suppress the denaturation of extracellular vesicles, the pH is preferably 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more. To improve the adsorption rate of extracellular vesicles, the pH is preferably less than 7, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[0121] In the pH adjustment step, inorganic salts such as sodium chloride and calcium chloride, and chaotropic salts such as guanidine thiocyanate and urea may be added as needed.

[0122] <Adsorption process> In the adsorption step, a liquid containing extracellular vesicles is mixed with component (A) and component (B), and the extracellular vesicles are adsorbed onto the surface of component (A). The adsorption rate of extracellular vesicles to the surface of the solid support can be increased by carrying out the adsorption step in the presence of component (B), which contains carbonate ions, phosphate ions, and calcium ions.

[0123] Component (A) and component (B) may be mixed in a solid state with a liquid containing extracellular vesicles, or an aqueous solution containing component (A) and / or component (B) may be prepared in advance and mixed in the aqueous solution state with a liquid containing extracellular vesicles.

[0124] Contact between a liquid containing extracellular vesicles and a solid phase carrier can be achieved, for example, by adding the solid phase carrier to the liquid, stirring, and dispersing the solid phase carrier in the liquid. The adsorption time is not particularly limited, but may be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, in order to allow the extracellular vesicles in the liquid containing the extracellular vesicles to sufficiently contact the surface of the solid phase carrier, and may be 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more. The adsorption time may be 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, and may be 30 minutes or less, 20 minutes or less, or 10 minutes or less, in order to facilitate suppression of denaturation of the extracellular vesicles.

[0125] In the adsorption step, to further increase the adsorption rate of extracellular vesicles, the amount of solid phase carrier added is preferably 1 mg or more, 2 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, or 50 mg or more per mL of liquid containing extracellular vesicles. To suppress the incorporation of impurities derived from the solid phase carrier, the amount of solid phase carrier added is preferably 100 mg or less, 75 mg or less, 50 mg or less, 30 mg or less, 20 mg or less, 10 mg or less, 5 mg or less, 3 mg or less, or 2 mg or less per mL of liquid containing extracellular vesicles.

[0126] The total amount of carbonate, phosphate, and calcium salt added is preferably 1 mg / L or more, more preferably 10 mg / L or more, 20 mg / L or more, 50 mg / L or more, 100 mg / L or more, 150 mg / L or more, 200 mg / L or more, 500 mg / L or more, 1000 mg / L or more, or 2000 mg / L, more preferably 1 mg / L or more, 2000 mg / L or more, more preferably 1 mg / L or more, 2000 mg / L or more, more preferably 10 mg / L or more, 2000 mg / L or more, 500 mg / L or more, 1000 mg / L or more, 150 mg / L or more, 200 mg / L or more, 500 mg / L or more, 1000 mg / L or more, 1000 mg / L or less, 8000 mg / L or less, 6000 mg / L or less, 4000 mg / L or less, 2000 mg / L or less, 1000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, or 200 mg / L or less, more preferably 10000 mg / L or less, more preferably 10000 mg / L or less, 8000 mg / L or less, 6000 mg / L or less, 400 mg / L or less, or 200 mg / L or less, more preferably 10000 mg / L or less, 800 mg / L or less, 6 ...

[0127] When the liquid containing extracellular vesicles is brought into contact with the component (B) in a solution state, the concentration of carbonate in the component (B) in a solution state is, based on the total amount of the liquid containing extracellular vesicles used and the amount of the component (B) in an aqueous solution state used, 50 mg / L or more, 80 mg / L or more, 100 mg / L or more, 500 mg / L or more, 1000 mg / L or more, 1500 mg / L or more, 20 ... It may be 500 mg / L or more, 3000 mg / L or more, or 3500 mg / L or more, and may be 500,000 mg / L or less, 400,000 mg / L or less, 300,000 mg / L or less, 200,000 mg / L or less, 100,000 mg / L or less, 50,000 mg / L or less, 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, or 4,000 mg / L or less.

[0128] The concentration of phosphate in the solution of component (B) when contacting the liquid containing extracellular vesicles with component (B) in the solution state may be 1 mg / L or more, 5 mg / L or more, 10 mg / L or more, 20 mg / L or more, 40 mg / L or more, 60 mg / L or more, 80 mg / L or more, 90 mg / L or more, or 100 mg / L or more, based on the total amount of the liquid containing extracellular vesicles used and the amount of component (B) in the aqueous solution state used, and may be 200 mg / L or more. The concentration may be 00mg / L or less, 15000mg / L or less, 12000mg / L or less, 10000mg / L or less, 8000mg / L or less, 6000mg / L or less, 4000mg / L or less, 2000mg / L or less, 1500mg / L or less, 1200mg / L or less, 1000mg / L or less, 800mg / L or less, 600mg / L or less, 400mg / L or less, 200mg / L or less, 150mg / L or less, or 120mg / L or less.

[0129] The concentration of calcium salt in component (B) in a solution state may be 1 mg / L or more, 5 mg / L or more, 10 mg / L or more, 20 mg / L or more, 40 mg / L or more, 60 mg / L or more, 80 mg / L or more, 90 mg / L or more, 100 mg / L or more, 140 mg / L or more, 160 mg / L or more, or 180 mg / L or more, based on the total volume of component (B) in a solution state and the sum of the amount of liquid containing extracellular vesicles used and the amount of component (B) in an aqueous solution state used. mg / L or less, 25,000 mg / L or less, 20,000 mg / L or less, 15,000 mg / L or less, 12,000 mg / L or less, 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, 4,000 mg / L or less, 2,500 mg / L or less, 2,000 mg / L or less, 1,500 mg / L or less, 1,200 mg / L or less, 1,000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, 300 mg / L or less, or 250 mg / L or less.

[0130] <Separation process> In the separation step, component (A) adsorbed with extracellular vesicles is separated from the liquid. The separation method is not particularly limited, but separation using a magnet is preferred for ease of operation. The separation method may be a combination of magnetic separation and, if necessary, centrifugation, filtration, natural sedimentation, etc.

[0131] <Cleaning process> In the washing step, the component (A) with adsorbed extracellular vesicles obtained in the separation step is washed. Examples of liquids (washing liquids) for washing the component (A) include buffer solutions, component (B) in aqueous solution, and acidic or alkaline liquids. Examples of buffer solutions include acetate buffer, phosphate buffer, citrate buffer, Tris-HCl buffer, and HEPES buffer. The pH of the buffer used as the washing liquid may be 5.0 to 10.0, 6.0 to 10.0, greater than 7.0 and less than 10.0, or 8.0 to 10.0. The acidic or alkaline liquid may be a liquid within the above pH range.

[0132] The washing method is not particularly limited, and examples thereof include a method in which component (A) is mixed with a washing liquid and the washing liquid is stirred. The temperature of the washing liquid in the washing step may be 10 to 40°C or 15 to 25°C. The washing time in the washing step (such as the stirring time of the washing liquid) can be appropriately selected depending on the type of washing liquid, the temperature of the washing liquid, etc. The washing time may be, for example, 1 minute or more, or 3 minutes or more, or 30 minutes or less, 15 minutes or less, or 10 minutes or less.

[0133] The separation step and washing step may be repeated in this order to repeatedly wash component (A) to which extracellular vesicles have been adsorbed with the washing solution. By repeatedly performing the separation step and washing step, more residues other than extracellular vesicles adsorbed to component (A) can be removed.

[0134] <Desorption process> In the desorption step, the extracellular vesicles are desorbed from component (A) to which they have been adsorbed. Methods for desorbing extracellular vesicles from component (A) include contacting component (A) to which the extracellular vesicles have been adsorbed with a liquid (desorption liquid) for desorbing the extracellular vesicles.

[0135] The desorption liquid is preferably a liquid containing a chelating agent, and examples of the chelating agent that can be used include those exemplified in the extracellular vesicle purification kit.

[0136] In order to promote the desorption of extracellular vesicles, the content of the chelating agent in the desorption solution is preferably 0.01 mmol / L or more, 0.1 mmol / L or more, 0.3 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 2 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 50 mmol / L or more, or 100 mmol / L or more, based on the total volume of the desorption solution. In order to promote the desorption of extracellular vesicles and suppress the denaturation of extracellular vesicles, the content of the chelating agent in the desorption solution is preferably 1000 mmol / L or less, 500 mmol / L or less, 300 mmol / L or less, 150 mmol / L or less, 80 mmol / L or less, 60 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 15 mmol / L or less, 8 mmol / L or less, 6 mmol / L or less, 3 mmol / L or less, 1.5 mmol / L or less, 0.8 mmol / L or less, 0.4 mmol / L or less, 0.2 mmol / L or less, or 0.05 mmol / L or less, based on the total amount of the desorption solution.

[0137] The pH of the desorption liquid may be 6 or higher, and in order to further increase the desorption rate of extracellular vesicles, it is preferably 7 or higher, 8 or higher, 9 or higher, 10 or higher, 11 or higher, 12 or higher, 13 or higher, or 14 or higher. In order to suppress the denaturation of extracellular vesicles, the pH of the liquid used in the desorption step is preferably 14 or lower, 13 or lower, 12 or lower, 11 or lower, 10 or lower, 9 or lower, or 8 or lower.

[0138] The desorption liquid may be an alkaline liquid, which makes it easier to desorb extracellular vesicles from the solid phase carrier.

[0139] The method for adjusting the pH of the desorption liquid is not particularly limited, and examples thereof include adding a base such as sodium hydroxide, sodium phosphate, sodium citrate, sodium carbonate, or sodium bicarbonate.

[0140] The desorption time is not particularly limited, but may be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more to sufficiently desorb the extracellular vesicles in the liquid from the surface of the solid phase carrier, or 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, 9 hours or more, or 11 hours or more. The desorption time may be 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or 30 minutes or less, 20 minutes or less, or 10 minutes or less to suppress denaturation of the extracellular vesicles.

[0141] In recent years, it has been reported that small extracellular vesicles (exosomes) with particle sizes of approximately 50 to 150 nm function as markers for cancer and other diseases, but conventional methods for purifying extracellular vesicles have not been able to recover extracellular vesicles with a specific particle size distribution. According to the method of this embodiment, in the desorption step, the extracellular vesicles are desorbed from the surface of the solid support to which they are adsorbed, and the liquid containing the desorbed extracellular vesicles is recovered (recovery operation) by repeating this operation two or more times, making it possible to recover extracellular vesicles with a specific particle size range.

[0142] In the desorption step, when the recovery operation is repeated two or more times, it is preferable to change the desorption time for each recovery operation. The desorption time for each recovery operation can be selected arbitrarily from the desorption times described above. For example, by setting the desorption time for the first recovery operation to 1 minute to 1 hour and the desorption time for the second recovery operation to 1 hour to 72 hours, it is possible to separate extracellular vesicles with a particle size of more than 150 nm from extracellular vesicles with a particle size of around 50 to 150 nm.

[0143] The method for purifying extracellular vesicles may further include a step of removing proteins from the liquid containing extracellular vesicles (protein removal step) before the pH adjustment step. By removing proteins in advance, it is possible to suppress adsorption of contaminants to the solid phase carrier, thereby further increasing the adsorption rate of extracellular vesicles. Examples of protein removal methods include methods of precipitating and removing proteins using salting out, polymer flocculants, or organic solvents, and methods of adding a protein adsorbent.

[0144] The extracellular vesicle purification method is preferably capable of adsorbing 30% or more of the extracellular vesicles contained in a serum-free medium sample, more preferably 50% or more, particularly preferably 70% or more, and most preferably 80% or more. For a serum-containing sample, it is preferably capable of adsorbing 20% ​​or more of the extracellular vesicles contained in the sample, more preferably 40% or more, particularly preferably 60% or more, and most preferably 70% or more.

[0145] In the method for purifying extracellular vesicles, for serum-free medium samples, it is preferable that 10% or more of the extracellular vesicles captured by the solid phase carrier can be detached, more preferably 30% or more, particularly preferably 50% or more, and most preferably 70% or more.For serum-containing samples, it is preferable that 10% or more of the extracellular vesicles captured by the solid phase carrier can be detached, more preferably 20% or more, particularly preferably 40% or more, and most preferably 60% or more. [Example]

[0146] The present invention will be described in detail below with reference to embodiments for carrying out the present invention. However, these are merely examples for explaining the present invention and are not intended to limit the present invention to the following content. Furthermore, the present invention can be practiced with appropriate modifications within the scope of the gist of the present invention. Unless otherwise specified, commercially available reagents were used. "Room temperature" means 20±10°C.

[0147] [Evaluation of extracellular vesicle adsorption rate] Cells were cultured for three days in serum-free medium or medium supplemented with 10% exosome-depleted serum, and 0.5 mL of the culture medium (culture supernatant; liquid containing extracellular vesicles) was mixed with 0.5 mL of an aqueous solution of the adsorption promoter. 10 mg of solid support was added to this mixture and stirred at room temperature for 1 hour. The supernatant, after removing the solid support from the solution, was used as the measurement sample.

[0148] The concentration of extracellular vesicles was measured by evaluation using Qubit (Thermo Fisher Scientific) with microRNA as the measurement target, and the adsorption rate was calculated using the following formula. (Concentration before adding solid phase carrier - concentration after adding solid phase carrier) x 100 / concentration before adding solid phase carrier [%]

[0149] [Evaluation of extracellular vesicle loss rate during washing] Extracellular vesicles were adsorbed onto the surface of the solid support using the same procedure as used to evaluate the extracellular vesicle adsorption rate. After separating the solid support with adsorbed extracellular vesicles, 10 mmol / L Tris-HCl buffer (pH 9) was added and stirred at room temperature for 5 minutes. The supernatant liquid obtained by removing the solid support from the solution was used as the measurement sample. As with the evaluation of the extracellular vesicle adsorption rate, the extracellular vesicle concentration was determined using the microRNA concentration as the target, and the washing loss rate was calculated using the following formula. (Amount of extracellular vesicles in the solution) x 100 / Concentration before adding the solid support [%]

[0150] [Evaluation of extracellular vesicle recovery rate] Using the same procedures as those used to evaluate the extracellular vesicle adsorption rate and washing loss rate, extracellular vesicles were adsorbed onto the surface of the solid support and then washed. After separating the solid support with adsorbed extracellular vesicles, a 1 mmol / L EDTA solution was added and stirred at room temperature for 1 hour. The supernatant, after removing the solid support from the solution, was used as the measurement sample. As with the evaluation of the extracellular vesicle adsorption rate, the extracellular vesicle concentration was calculated using the microRNA concentration as the target, and the desorption rate was calculated using the following formula. (Amount of extracellular vesicles in the solution) x 100 / Concentration before adding the solid support [%]

[0151] [Test 1 to confirm the effect of using a solid support having a metal oxide on its surface] [Example 1-1] (Synthesis of magnetic particles) Process (i) (fine magnetic material adsorption) 1 g of polydivinylbenzene particles (particle diameter 2.2 μm) was dispersed in 50 mL of 0.1 M sodium chloride pure water at room temperature at 100 rpm. 1.2 g of a magnetic material (material: magnetite, particle diameter: 10 nm, product name: EMG607, manufactured by Ferrotec Corporation) bearing a cationic dispersant on its surface was added to this particle dispersion, and the mixture was allowed to react at room temperature at 100 rpm for 2 hours. The solid content (particles) was filtered off and washed with pure water.

[0152] Step (ii) (Drying and High-Velocity Airflow Treatment) The particles washed with pure water were dried at 50°C for 15 hours to obtain 2.18 g of magnetized dried particles. These magnetized particles were placed in a hybridization system NHS-0 (manufactured by Nara Machinery Co., Ltd.) and rotated at a speed of 11,300 min -1 The treatment was carried out for 5 minutes at an average temperature of 35°C during treatment.

[0153] Step (iii) (Metal oxide coating on magnetized particles) 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, 0.4 g of tetraethyl orthosilicate was added, and the mixture was stirred at room temperature at 180 rpm. 0.35 mL of 25% aqueous ammonia was added, and the mixture was reacted at 60°C and 180 rpm for 4 hours. The particles were washed with methanol and dried under reduced pressure. The cross-sectional shape of the particles was confirmed using a transmission electron microscope, and the thickness of the metal oxide layer was approximately 0.02 to 0.05 μm.

[0154] (adsorption promoter) The following procedure was performed to obtain final concentrations of 3700 mg / L sodium bicarbonate, 109 mg / L disodium hydrogen phosphate, and 200 mg / L calcium chloride. First, stock solutions of each component were prepared at 10 times the final concentration. Next, these solutions were mixed and diluted with pure water to adjust the concentration. This aqueous solution was used as an adsorption promoter and mixed with the liquid containing extracellular vesicles in a 1:1 volume ratio.

[0155] (Evaluation results) The adsorption rate of extracellular vesicles was 93%, which was significantly high. The washing loss rate was 11%, which was low. The recovery rate was 90%, which was significantly high.

[0156] [Example 1-2] The evaluation was carried out in the same manner as in Example 1-1, except that sodium dihydrogen phosphate was used instead of disodium hydrogen phosphate as the adsorption promoter in Example 1-1.

[0157] (Evaluation results) The adsorption rate of extracellular vesicles was 83%, which was high. The washing loss rate was 4%, which was low. The recovery rate was 42%, which was high.

[0158] [Examples 1-3] The evaluation was carried out in the same manner as in Example 1-1, except that sodium bicarbonate was not used as the adsorption promoter in Example 1-1.

[0159] (Evaluation results) The adsorption rate of extracellular vesicles was 64%, and the recovery rate was 29%.

[0160] [Examples 1-4] The evaluation was carried out in the same manner as in Example 1-1, except that disodium hydrogen phosphate was not used as the adsorption promoter in Example 1-1.

[0161] (Evaluation results) The adsorption rate of extracellular vesicles was 68%, and the recovery rate was 26%.

[0162] [Examples 1-5] The evaluation was carried out in the same manner as in Example 1, except that calcium chloride was not used as the adsorption promoter in Example 1.

[0163] (Evaluation results) The adsorption rate of extracellular vesicles was 59%, and the recovery rate was 39%.

[0164] [Examples 1-6] The evaluation was carried out in the same manner as in Example 1-1, except that sodium chloride was used instead of calcium chloride in the adsorption promoter of Example 1-1.

[0165] (Evaluation results) The adsorption rate of extracellular vesicles was 38%, and the recovery rate was 13%.

[0166] [Comparative Example 1-1] The adsorption promoter of Example 1 was not used, and pure water was used instead. Otherwise, the evaluation was carried out in the same manner as in Example 1.

[0167] (Evaluation results) The adsorption rate of extracellular vesicles was 53%, the washing loss rate was 38%, and the recovery rate was 11%.

[0168] [Comparative Example 1-2] The evaluation was carried out in the same manner as in Example 1, except that disodium hydrogen phosphate and calcium chloride, which were used as adsorption promoters in Example 1, were not used.

[0169] (Evaluation results) The adsorption rate of extracellular vesicles was 46%. The recovery rate was low at 9%.

[0170] [Comparative Example 1-3] The evaluation was carried out in the same manner as in Example 1, except that sodium bicarbonate and calcium chloride were not used in the adsorption promoter of Example 1.

[0171] (Evaluation results) The adsorption rate of extracellular vesicles was 62%. The recovery rate was 0%, which was low.

[0172] [Comparative Example 1-4] The evaluation was carried out in the same manner as in Example 1-1, except that sodium bicarbonate and disodium hydrogen phosphate were not used as the adsorption promoters in Example 1-1.

[0173] (Evaluation results) The adsorption rate of extracellular vesicles was 63%. The recovery rate was 0%, which was low.

[0174] [Examples 2-1 to 2-26] The evaluation was carried out in the same manner as in Example 1-1, except that the amounts of carbonate, phosphate, and calcium salt used were changed as shown in the table.

[0175] [Test 2 to confirm the effect of using a solid support having a metal oxide on its surface] [Example 3-1] Magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1-1, and the following step (iii) was further carried out.

[0176] Step (iii) (Metal oxide coating on magnetized particles) 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, 0.8 g of tetraethyl orthotitanate was added, and the mixture was stirred at room temperature at 180 rpm. 0.4 mL of 25% aqueous ammonia was added, and the mixture was reacted at 60°C and 180 rpm for 4 hours. The particles were washed with methanol and dried under reduced pressure. Using the above method, magnetic particles with a micromagnetic material content of 0.59 g / g per 1 g of magnetic particles were obtained.

[0177] (Evaluation results) The adsorption rate of extracellular vesicles was 89%, which was high. The washing loss rate was 8%, which was low. The recovery rate was 42%, which was high.

[0178] [Example 3-2] Magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 1-1, and the following step (iii) was further carried out.

[0179] Step (iii) (Metal oxide coating on magnetized particles) A coating layer consisting of indium oxide and indium tin oxide was formed on the surface of the magnetized particles by sputtering them for 8 hours using a target mixture of 95% by mass of indium oxide and 5% by mass of tin oxide. The sputtering was performed at a power of 1000 W and a gas pressure of 1.5 Pa in the presence of a mixed gas of argon and oxygen. The cross-sectional shape of the particles was confirmed using a transmission electron microscope, and the thickness of the metal oxide layer was approximately 0.06 to 0.11 μm.

[0180] (Evaluation results) The adsorption rate of extracellular vesicles was 88%, which was high. The washing loss rate was 2%, which was low. The recovery rate was 65%, which was high.

[0181] [Test to confirm the effect of changing the type of solid phase carrier] [Example 4-1] A column packed with commercially available silica gel particles was used, and the other procedures were the same as in Example 1-1, and evaluation was carried out.

[0182] (Evaluation results) The adsorption rate of extracellular vesicles was 98%, which was high. The washing loss rate was 13%, which was low. The recovery rate was 85%, which was high.

[0183] [Example 4-2] (Preparation of well plates) A polycarbonate film coated with silicon dioxide by sputtering was attached to the bottom of a bottomless 24-well plate.

[0184] (Evaluation method) 200 μL of a liquid containing extracellular vesicles was used per well of a 24-well plate. Mixing was performed by shaking, and the evaluation was otherwise carried out in the same manner as in Example 1-1.

[0185] (Evaluation results) The adsorption rate of extracellular vesicles was 78%, which was high. The washing loss rate was 2%, which was low. The recovery rate was 41%, which was high.

[0186] [Test to confirm the effect of using a solid phase carrier having a polymer on its surface] [Example 5-1] (method) Magnetic particles were synthesized in the same manner as in Example 2-3, except that the metal oxide coating in Example 1-1 was not performed and the surface was coated with a polymer by the following procedure. These were designated as magnetic particles of Example 5-1.

[0187] (Polymer coating on magnetic particles) 10 g of magnetic particles were dispersed in 30 mL of methanol, and 4 g of 3-methacryloxypropyltrimethoxysilane and 4 mL of 25% aqueous ammonia were added. The mixture was reacted at 60°C for 3 hours at 180 rpm. The mixture was washed with methanol and pure water and dried under reduced pressure. 1 g of these particles was dispersed in 9 mL of pure water, and 0.2 g of cyclohexyl methacrylate, 0.035 g of methacrylic acid, 0.3 g of polyoxyethylene lauryl ether, and 0.005 g of initiator were added. After degassing, the mixture was reacted at 80°C for 2 hours under a nitrogen atmosphere. The mixture was washed with pure water and methanol and dried under reduced pressure.

[0188] (Evaluation results) The adsorption rate of extracellular vesicles was 82%, which was high. The washing loss rate was 12%, which was low. The recovery rate was 68%, which was high.

[0189] Magnetic particles of Comparative Example 5-1 were prepared using pure water instead of the adsorption promoter used in Example 5-1. Otherwise, evaluation was carried out using the same procedures as in Example 5-1.

[0190] (Evaluation results) A comparison between Example 5-1 and Comparative Example 5-1 confirmed that even when the solid phase carrier has a polymer on its surface, the adsorption rate, cleaning loss rate, and recovery rate can be improved by using an adsorption promoter.

[0191] Regarding the results for "Extracellular vesicles (measured by CD63)" in the table, "-" indicates that the results have not been evaluated.

[0192] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Explanation of symbols]

[0193] 1...core portion, 2...micromagnetic body, 3...metal oxide, 4...surface layer, 10...solid phase carrier (magnetic particle)

Claims

1. An extracellular vesicle purification kit comprising the following (A) and (B): (A) Solid support. (B) A component containing two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions.

2. The extracellular vesicle purification kit of claim 1, further comprising the following (C): (C) a chelating agent.

3. The kit for extracellular vesicle purification according to claim 1 or 2, wherein the component (B) is a component containing carbonate ions, phosphate ions, and calcium ions.

4. The kit for extracellular vesicle purification according to claim 1 or 2, wherein the component (B) comprises a carbonate, a phosphate, and a calcium salt.

5. the carbonate is sodium bicarbonate, the phosphate is sodium dihydrogen phosphate or disodium hydrogen phosphate, The extracellular vesicle purification kit of claim 4, wherein the calcium salt is calcium chloride.

6. The kit for extracellular vesicle purification according to claim 1 or 2, wherein the solid support has a metal oxide on its surface.

7. The extracellular vesicle purification kit of claim 6, wherein the metal oxide is one or more components selected from the group consisting of zinc oxide, titanium oxide, silicon dioxide, nickel oxide, yttria oxide, tin oxide, indium oxide, and indium tin oxide.

8. The kit for purifying extracellular vesicles according to claim 1 or 2, wherein the solid phase carrier is a magnetic particle having a metal oxide on its surface.

9. The kit for extracellular vesicle purification according to claim 1 or 2, wherein the solid phase carrier has a non-adhesive layer for extracellular vesicles on its surface.

10. A method for purifying extracellular vesicles using the extracellular vesicle purification kit according to claim 1 or 2, comprising: an adsorption step of mixing a liquid containing the extracellular vesicles with the component (A) and the component (B) to adsorb the extracellular vesicles onto the surface of the component (A); a separation step of separating the component (A) adsorbed by the extracellular vesicles from the liquid; a washing step of washing the component (A) to which the extracellular vesicles have been adsorbed; A desorption step of desorbing the extracellular vesicles from the component (A) to which the extracellular vesicles have been adsorbed.

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