Extracellular vesicle purification kit and method for purifying extracellular vesicle

Abstract

The present disclosure pertains to an extracellular vesicle purification kit that contains a component that does not dissolve in pure water but dissolves in a chelating agent aqueous solution.

Description

Extracellular vesicle purification kit and extracellular vesicle purification method

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

[0002] In recent years, there has been a demand for a technology for recovering vesicles with a heterogeneous lipid bilayer structure (hereinafter referred to as "extracellular vesicles") released by cells from a sample. Regarding a technology for purifying extracellular vesicles, Patent Document 1 discloses a technology that uses a device in which zinc oxide protrusions are formed within a flow channel.

[0003] Patent No. 6606786

[0004] An object of the present invention is to provide an extracellular vesicle purification kit with an improved recovery rate of extracellular vesicles. Another object of the present invention is to provide a method for purifying extracellular vesicles using the above-mentioned extracellular vesicle purification kit. Another object of the present invention is to provide a method for producing a material for extracellular vesicle purification that can improve the recovery rate of extracellular vesicles.

[0005] Each aspect of the present invention is described below. [1] An extracellular vesicle purification kit comprising a component that is insoluble in pure water but soluble in an aqueous chelating agent solution. [2] The extracellular vesicle purification kit according to [1], wherein the component comprises carbon, phosphorus, and oxygen. [3] The extracellular vesicle purification kit according to [2], wherein the number of moles of carbon in the component is 5.0 moles or less per mole of phosphorus in the component. [4] The extracellular vesicle purification kit according to [2] or [3], wherein the number of moles of oxygen in the component is 1.00 to 20.00 moles per mole of phosphorus in the component. [5] The extracellular vesicle purification kit according to any of [2] to [4], wherein the component further comprises calcium. [6] The extracellular vesicle purification kit according to any of [2] to [5], wherein the number of moles of calcium in the component is 1.00 to 10.00 moles per mole of phosphorus in the component. [7] The extracellular vesicle purification kit according to any one of [2] to [6], wherein the component further comprises hydrogen, and the number of moles of hydrogen in the component is 0.1 to 10.0 moles per mole of phosphorus in the component. [8] The extracellular vesicle purification kit according to any one of [1] to [7], wherein the component has a peak with the highest intensity in an X-ray diffraction spectrum within a 2θ value range of 30° to 35°. [9] The extracellular vesicle purification kit according to any one of [1] to [8], wherein the full width at half maximum of the peak with the highest intensity in the X-ray diffraction spectrum of the component is within a range of 0.2° to 5.5°.

[10] The extracellular vesicle purification kit according to any one of [1] to [9], wherein the component contains phosphate ions and calcium ions as constituent ions.

[11] The extracellular vesicle purification kit according to any one of [1] to

[10] , wherein the component is a reaction product of a carbonate, a phosphate, and a calcium salt.

[12] The kit for extracellular vesicle purification according to any one of [1] to

[11] , wherein the ratio of the mass of the component in a dry state to the volume of the component in a swollen state is 10 to 100 g / L.

[13] The kit for extracellular vesicle purification according to any one of [1] to

[12] , wherein the haze of the liquid in which the component is dispersed is 70 to 100%.

[14] The extracellular vesicle purification kit according to any one of [1] to

[13] , wherein the component is a cluster-like secondary particle formed by association of primary particles, and the ratio of the average diameter of the secondary particles to the average diameter of the primary particles is 10 to 1000.

[15] The extracellular vesicle purification kit according to any one of [1] to

[14] , wherein the component has, in an X-ray diffraction spectrum, peaks at 2θ between 30° and 35° and between 2θ between 28° and 30°, and wherein the ratio of the intensity of the peak at 2θ between 28° and 30° to the intensity of the peak at 2θ between 30° and 35° is 0 to 0.13.

[16] The extracellular vesicle purification kit according to any one of [1] to

[15] , further comprising a chelating agent.

[17] The extracellular vesicle purification kit according to any one of [1] to

[16] , comprising a solid phase support comprising the component at least on its surface.

[18] The extracellular vesicle purification kit according to

[17] , wherein the solid phase carrier is a magnetic particle.

[19] The extracellular vesicle purification kit according to any of [1] to

[18] , comprising a solid phase carrier comprising: a core portion; an intermediate layer comprising an extracellular vesicle non-adhesive layer and covering at least a portion of the surface of the core portion; and a surface layer comprising the component and covering at least a portion of the surface of the intermediate layer.

[20] A method for purifying extracellular vesicles using the extracellular vesicle purification kit according to any of [1] to

[19] , comprising: an adsorption step of contacting a liquid containing the extracellular vesicles with the component to adsorb the extracellular vesicles to the component; a separation step of separating the component adsorbed by the extracellular vesicles from the liquid; a washing step of washing the component adsorbed by the extracellular vesicles; and a desorption step of detaching the extracellular vesicles from the component adsorbed by the extracellular vesicles.

[21] A method for producing a material for extracellular vesicle purification, comprising the step of reacting a carbonate, a phosphate, and a calcium salt in an aqueous solution to obtain the material for extracellular vesicle purification as a white precipitate.

[22] A kit for extracellular vesicle purification, comprising 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.

[23] The extracellular vesicle purification kit according to

[22] , further comprising a chelating agent.

[24] The extracellular vesicle purification kit according to

[22] or

[23] , wherein the component (B) is a component containing carbonate ions, phosphate ions, and calcium ions.

[25] The extracellular vesicle purification kit according to any of

[22] to

[24] , wherein the component (B) is a component containing a carbonate salt, a phosphate salt, and a calcium salt.

[26] The extracellular vesicle purification kit according to any of

[22] to

[25] , wherein the carbonate salt is sodium bicarbonate, the phosphate salt is sodium dihydrogen phosphate or disodium hydrogen phosphate, and the calcium salt is calcium chloride.

[27] The extracellular vesicle purification kit according to any of

[22] to

[26] , wherein the solid phase support has a metal oxide on its surface.

[28] The extracellular vesicle purification kit according to

[27] , 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.

[29] The extracellular vesicle purification kit according to any of

[22] to

[28] , wherein the solid phase carrier is magnetic particles having a metal oxide on the surface.

[30] The extracellular vesicle purification kit according to any of

[22] to

[29] , wherein the solid phase carrier has a non-adhesive layer for extracellular vesicles on the surface.

[31] A method for purifying extracellular vesicles using the extracellular vesicle purification kit according to any one of

[22] to

[31] , 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 onto 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.

[0006] 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. According to the present invention, it is possible to provide a method for producing a material for extracellular vesicle purification that can improve the recovery rate of extracellular vesicles.

[0007] Fig. 1 is a schematic diagram showing an example of a solid phase carrier according to one embodiment of the present invention. Fig. 2 is a schematic diagram showing an example of a solid phase carrier according to another embodiment of the present invention. Fig. 3 is a schematic diagram showing an example of a solid phase carrier according to another embodiment of the present invention.

[0008] 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.

[0009] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​written before and after "to" as the minimum and maximum values, respectively. Furthermore, unless specifically stated otherwise, the units of the numerical values ​​written before and after "to" are the same. In the numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in a certain stage may be replaced with the upper limit or lower limit of a numerical range in another stage. Furthermore, in the numerical ranges described in this specification, the upper limit or lower limit of the numerical range may be replaced with a value shown in the examples. Furthermore, the upper limit and lower limit values ​​individually described can be combined in any way. In this specification, "(meth)acrylate" means at least one of acrylate and its corresponding methacrylate.

[0010] [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, breast milk, and the like.

[0011] [First embodiment: component insoluble in pure water but soluble in a chelating agent aqueous solution] The extracellular vesicle purification kit according to the first embodiment includes a component (hereinafter also referred to as "component a") that is insoluble in pure water but soluble in a chelating agent aqueous solution. Component a is a component that is capable of adsorbing extracellular vesicles and is capable of detaching (desorbing) the adsorbed extracellular vesicles upon contact with a chelating agent. Component a can also be referred to as a material for extracellular vesicle purification. Component a may be one type of compound or a mixture of two or more types of compounds. Component a may be in a solid form. Examples of solid forms include a plate, a film, and particles.

[0012] When component a is a component that is insoluble in pure water but soluble in an aqueous chelating agent solution, extracellular vesicles in a sample containing extracellular vesicles can be adsorbed to component a, and then part or all of component a can be dissolved in an aqueous chelating agent solution to recover the extracellular vesicles. Purification of extracellular vesicles using the adsorption / desorption phenomenon is usually performed by removing the extracellular vesicles from the surface of the carrier without dissolving the carrier to which the extracellular vesicles are adsorbed. In such a method, some of the extracellular vesicles may remain on the surface of the carrier. On the other hand, dissolving component a in an aqueous chelating agent solution allows all of the extracellular vesicles adsorbed to component a to be recovered, resulting in excellent recovery rates of extracellular vesicles and reproducibility of purification. Furthermore, dissolving all of component a eliminates the need to remove the carrier, resulting in excellent workability and throughput.

[0013] In this specification, whether a certain component is insoluble or soluble can be determined by the following method in accordance with JIS K8001 General Rules for Testing Reagents.

[0014] A certain amount of a component is powdered (if the sample is solid) and placed in a solvent. The component is vigorously shaken for 30 seconds every 5 minutes at 20°C ± 5°C. Dissolution is defined as the volume (mL) of solvent required for dissolution within 30 minutes. If the amount of solvent required to completely dissolve 1 g or 1 mL of the component is 1 L or more, the component is considered insoluble. If the amount of solvent required to completely dissolve 1 g or 1 mL of the component is less than 1 L, the component is considered soluble.

[0015] When the solvent is pure water, distilled water is used. The amount of distilled water required to dissolve all of 1 g or 1 mL of component a may be 1 L or more, or 2 L or more.

[0016] The amount (g) of component a dissolved in 1 L of distilled water may be, for example, less than 10 g / L, 5 g / L or less, or 1 g / L or less.

[0017] When the solvent is an aqueous chelating agent solution, the aqueous chelating agent solution may be, for example, a 500 mM aqueous ethylenediaminetetraacetic acid solution. A 500 mM aqueous ethylenediaminetetraacetic acid solution can be prepared by dissolving ethylenediaminetetraacetic acid in distilled water to a concentration of 500 mM. The amount of the aqueous chelating agent solution required to dissolve all 1 g or 1 mL of component a is less than 1 L, and may be 0.5 L or less, 0.3 L or less, 0.1 L or less, 10 mL or less, or 1 mL or less.

[0018] The amount (g / L) of component a dissolved in 1 L of the chelating agent aqueous solution may be 1 g / L or more, 3 g / L or more, 5 g / L or more, or 10 g / L or more. The amount (g / L) of component a dissolved in 1 L of the chelating agent aqueous solution may be 100 g / L or less, 50 g / L or less, 20 g / L or less, 8 g / L or less, 4 g / L or less, or 2 g / L or less.

[0019] Component a may contain at least one element selected from the group consisting of carbon (C), phosphorus (P), and oxygen (O) as a constituent element. Component a may contain at least phosphorus (P) as a constituent element, or may contain at least phosphorus (P) and oxygen (O) as constituent elements, or may contain carbon (C), phosphorus (P), and oxygen (O) as constituent elements.

[0020] The number of moles of carbon element in component a may be 0.00 to 5.0 moles, or 0.00 to 1.0 mole, per mole of phosphorus element in component a.

[0021] The lower limit of the number of moles of carbon element may be 0.00 moles or more, 0.04 moles or more, 0.05 moles or more, 0.10 moles or more, 0.15 moles or more, 0.20 moles or more, 0.25 moles or more, 0.30 moles or more, 0.35 moles or more, 0.40 moles or more, 0.42 moles or more, 0.44 moles or more, 0.46 moles or more, 0.48 moles or more, 0.50 moles or more, 1.00 moles or more, 2.00 moles or more, 3.00 moles or more, or 4.00 moles or more, relative to 1 mole of phosphorus element in component a.

[0022] When component a contains carbon (for example, when the number of moles of carbon in component a is 0.04 moles or more per mole of phosphorus in component a), even when an aqueous chelating agent solution with a low concentration of the chelating agent that does not completely dissolve component a is added, it is possible to suppress readsorption of extracellular vesicles to component a and increase the recovery rate of extracellular vesicles. This allows the amount of liquid used to desorb extracellular vesicles to be reduced, allowing extracellular vesicles to be concentrated to a high concentration, or the concentration of the chelating agent in the liquid from which extracellular vesicles have been recovered to be reduced.

[0023] When component a contains carbon (for example, when the number of moles of carbon in component a is 0.04 moles or more per mole of phosphorus in component a), the bulk density of component a in a wet state can be further improved. As used herein, "bulk density in a wet state" refers to the ratio of the mass of component a in a dry state to the volume of component a in a swollen state (dry mass of component a / swollen volume). By improving the bulk density, extracellular vesicles can be adsorbed by simply adding a small volume of component a, allowing processing in a small reaction vessel.

[0024] When component a contains carbon elements (for example, when the number of moles of carbon elements in component a is 0.04 moles or more per mole of phosphorus element in component a), the transparency of component a can be reduced, and when component a and a liquid are separated by centrifugation or the like, the interface between component a and the supernatant liquid can be easily observed, making it easier to prevent operational errors such as erroneously discarding component a, and further improving workability.

[0025] The upper limit of the number of moles of carbon element may be 5.0 mols or less, 4.0 mols or less, 3.0 mols or less, 2.0 mols or less, 1.5 mols or less, 1.0 mols or less, 0.95 mols or less, 0.90 mols or less, 0.85 mols or less, 0.80 mols or less, 0.75 mols or less, 0.70 mols or less, 0.65 mols or less, 0.60 mols or less, 0.58 mols or less, 0.56 mols or less, 0.54 mols or less, 0.52 mols or less, 0.50 mols or less, 0.45 mols or less, 0.40 mols or less, 0.30 mols or less, 0.20 mols or less, 0.10 mols or less, or 0.05 mols or less, relative to 1 mole of phosphorus element in component a.

[0026] When component a contains a small amount of carbon element (for example, when the number of moles of carbon element in component a is 0.01 mole or less per mole of phosphorus element in component a), component a can adsorb extracellular vesicles even in the presence of a chelating agent, and exhibits a critical characteristic of the desorption amount relative to the chelating agent concentration, in which extracellular vesicles can be desorbed only when a certain amount or more of chelating agent is added. Furthermore, by adding component a again to a liquid containing extracellular vesicles desorbed using a chelating agent, the extracellular vesicles can be re-adsorbed to component a, and the purity of the extracellular vesicles can be increased by repeating the purification of extracellular vesicles using component a any number of times.

[0027] The number of moles of oxygen element may be 1.00 to 20.00 moles, 3.00 to 5.50 moles, or 4.10 to 5.00 moles per mole of phosphorus element in component a.

[0028] The lower limit of the number of moles of oxygen element may be 0.00 moles or more, 1.00 moles or more, 2.00 moles or more, 3.00 moles or more, 3.20 moles or more, 3.40 moles or more, 3.60 moles or more, 3.80 moles or more, 4.00 moles or more, 4.20 moles or more, 4.40 moles or more, 4.60 moles or more, 4.80 moles or more, 5.00 moles or more, 6.00 moles or more, 7.00 moles or more, 8.00 moles or more, 9.00 moles or more, 10.00 moles or more, 12.00 moles or more, 14.00 moles or more, 16.00 moles or more, or 18.00 moles or more, relative to 1 mole of phosphorus element in component a.

[0029] The upper limit of the number of moles of oxygen element may be 20.00 mols or less, 17.00 mols or less, 15.00 mols or less, 13.00 mols or less, 11.00 mols or less, 10.00 mols or less, 9.00 mols or less, 8.00 mols or less, 7.00 mols or less, 6.00 mols or less, 5.50 mols or less, 5.25 mols or less, 5.00 mols or less, 4.90 mols or less, 4.80 mols or less, 4.60 mols or less, 4.40 mols or less, 4.20 mols or less, 4.00 mols or less, 3.80 mols or less, 3.60 mols or less, or 3.50 mols or less, relative to 1 mole of phosphorus element in component a.

[0030] Component a may further contain calcium element (Ca). The number of moles of calcium element in component a may be 1.00 to 10.00 moles, 1.00 to 2.00 moles, or 1.10 to 1.60 moles per mole of phosphorus element in component a. The lower limit of the number of moles of calcium element in component a may be 1.00 moles or more, 1.05 moles or more, 1.10 moles or more, 1.15 moles or more, 1.20 moles or more, 1.25 moles or more, 1.30 moles or more, 1.35 moles or more, 1.40 moles or more, 1.45 moles or more, 1.50 moles or more, 1.55 moles or more, 2.00 moles or more, 3.00 moles or more, 4.00 moles or more, 5.00 moles or more, 6.00 moles or more, 7.00 moles or more, 8.00 moles or more, or 9.00 moles or more per mole of phosphorus element in component a. The upper limit of the number of moles of calcium element in component a may be 10.00 mols or less, 9.00 mols or less, 8.00 mols or less, 7.00 mols or less, 6.00 mols or less, 5.00 mols or less, 4.00 mols or less, 3.00 mols or less, 2.00 mols or less, 1.80 mols or less, 1.60 mols or less, 1.50 mols or less, 1.40 mols or less, or 1.35 mols or less.

[0031] Component a may further contain hydrogen element (H). The number of moles of hydrogen element in component a may be 0.1 to 10.0 moles, or 0.1 to 4.0 moles, relative to 1 mole of phosphorus element in component a. The lower limit of the number of moles of hydrogen element in component a may be 0.2 moles or more, 0.4 moles or more, 0.6 moles or more, 0.8 moles or more, 0.9 moles or more, 1.0 moles or more, 1.5 moles or more, 2.0 moles or more, 2.5 moles or more, 3.0 moles or more, 4.0 moles or more, 5.0 moles or more, 6.0 moles or more, 7.0 moles or more, 8.0 moles or more, or 9.0 moles or more, relative to 1 mole of phosphorus element in component a. The upper limit of the number of moles of hydrogen element in component a may be 10.0 mols or less, 9.0 mols or less, 8.0 mols or less, 7.0 mols or less, 6.0 mols or less, 5.0 mols or less, 4.0 mols or less, 3.6 mols or less, 3.2 mols or less, 2.8 mols or less, 2.7 mols or less, 2.4 mols or less, 2.0 mols or less, 1.6 mols or less, 1.2 mols or less, 1.0 mols or less, 0.8 mols or less, or 0.5 mols or less.

[0032] Component a may be, for example, a component in which the mole numbers of carbon, oxygen, hydrogen, and calcium per mole of phosphorus are the values ​​shown in Table 8 of the Examples section described below. Component a may be, for example, a component in which the mole numbers of carbon, oxygen, hydrogen, and calcium per mole of phosphorus are 0.48 mol, 4.89 mol, 2.61 mol, and 1.59 mol, respectively, a component in which the mole numbers of carbon, oxygen, hydrogen, and calcium per mole of phosphorus are 0.09 mol, 3.43 mol, 2.61 mol, and 1.59 mol, respectively, or a component in which the mole numbers of carbon, oxygen, hydrogen, and calcium per mole of phosphorus are 0.00 mol, 4.00 mol, 0.92 mol, and 1.30 mol, respectively.

[0033] The constituent elements of component a and their content ratios are measured by CHN elemental analysis for carbon and hydrogen, and by scanning electron microscope energy dispersive X-ray spectroscopy (SEM-EDS analysis) for phosphorus, oxygen and calcium.

[0034] The zeta potential of component a is suitable for specifically adsorbing extracellular vesicles, and therefore may be a negative value, such as -40 mV or more, -30 mV or more, -20 mV or more, -10 mV or more, or -5 mV or more. Furthermore, the zeta potential of component a may be 0 mV or less, -2 mV or less, -8 mV or less, -15 mV or less, -25 mV or less, or -35 mV or less. Since this further increases the adsorption strength of extracellular vesicles and further reduces loss during washing, the zeta potential of component a may be 0 mV or a positive value, such as 0 mV or more, 2 mV or more, 8 mV or more, 15 mV or more, 25 mV or more, or 35 mV or more. Furthermore, the zeta potential of component a may be 40 mV or less, 30 mV or less, 20 mV or less, 10 mV or less, or 5 mV or less. The zeta potential is measured using a solution having a pH in the range of 6 to 8.

[0035] The zeta potential of component a may be −40 to 40 mV, −40 to 30 mV, −40 to 20 mV, −40 to 10 mV, −35 to 5 mV, −30 to 0 mV, −25 to −5 mV, −25 to −10 mV, or −20 to −15 mV.

[0036] When a small amount of component a is added during purification of a trace amount of extracellular vesicles, component a is easily visible, so the dry mass / swelling volume (g / L) of component a is preferably 0.1 g / L or more, 0.5 g / L or more, 1 g / L or more, 1.5 g / L or more, 2 g / L or more, 2.5 g / L or more, 3 g / L or more, 3.5 g / L or more, 4 g / L or more, 4.5 g / L or more, 5 g / L or more, 5.5 g / L or more, 6 g / L or more, 6.5 g / L or more, 7 g / L or more, 7 g / L or more, 8 g / L or more, 9 g / L or more, 10 g / L or more, 11 g / L or more, 12 g / L or more, 13 g / L or more, 14 g / L or more, 15 g / L or more, 16 g / L or more, 17 g / L or more, 18 g / L or more, 19 g / L or more, 20 g / L or more, 21 g / L or more, 22 g / L or more, 23 g / L or more, 24 g / L or more, 25 g / L or more, 26 g / L or more, 27 g / L or more, 28 g / L or more, 29 g / L or more, 30 g / L or more, 31 g / L or more, 32 g / L or more, 33 g / L or more, 34 g / L or more, 35 g / L or more, 36 g / L or more, 37 g / L or more, 38 g / L or more, 39 g / L or more, 40 g / L or more, 41 g / L or more, 42 g / L or more, 43 g / L . 5g / L or more, 8g / L or more, 8g / L or more, 8.5g / L or more, 9g / L or more, 9.5g / L or more, 10g / L or more, 11g / L or more, 12g / L or more, 13g / L or more, 14g / L or more, 15g / L or more, 16g / L or more, 17g / L or more, 18g / L or more, 19g / L or more, 20g / L or more, 25g / L or more, 30g / L or more, 35g / L or more, 40g / L or more, 45g / L or more, or 50g / L or more. Furthermore, since extracellular vesicles can be adsorbed by simply adding a small volume of component a and processing can be performed in a small reaction vessel, the dry mass / swelling volume of component a may be 100 g / L or less, 90 g / L or less, 80 g / L or less, 70 g / L or less, 60 g / L or less, 50 g / L or less, 40 g / L or less, 30 g / L or less, 25 g / L or less, 20 g / L or less, 18 g / L or less, 16 g / L or less, 14 g / L or less, 12 g / L or less, 10 g / L or less, 9 g / L or less, 8 g / L or less, 7 g / L or less, 6 g / L or less, 5 g / L or less, 4 g / L or less, 3 g / L or less, 2 g / L or less, or 1 g / L or less.

[0037] The dry mass / swelling volume (g / L) of component a may be 0.1 to 100 g / L, 1 to 50 g / L, 1 to 30 g / L, 2 to 20 g / L, or 10 to 20 g / L.

[0038] The dry mass / swollen volume of component a can be calculated from the mass (g) of component a in a dry state / the volume (L) of component a in a swollen state, using the volume of component a in a swollen state (sedimentary layer) obtained by dispersing component a in pure water, centrifuging the dispersion at 5,000 g for 5 minutes, and removing all of the supernatant, and the mass of component a after drying the sedimentary layer.

[0039] The average diameter of component a is suitable for increasing dispersibility in liquid, and may be 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 8 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, 0.5 μm or less, or 0.1 μm or less. Furthermore, since the sedimentation rate by centrifugation can be further improved, the average diameter of component a may be 0.3 μm or more, 0.6 μm or more, 0.9 μm or more, 1.5 μm or more, 2.1 μm or more, 2.4 μm or more, 2.7 μm or more, 3.1 μm or more, 3.5 μm or more, 3.8 μm or more, 4.5 μm or more, 7 μm or more, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, or 90 μm or more.

[0040] The average diameter of component a may be 0.3 to 100 μm, 0.5 to 50 μm, 0.7 to 20 μm, 1 to 10 μm, or 1 to 5 μm.

[0041] In this specification, the average diameter is the number average particle diameter measured using a particle size distribution measuring device.

[0042] Since it is suitable for achieving both dispersibility in a liquid and an increase in the sedimentation rate by centrifugation, the particle sizes of component a that reach 10% when calculated from the smallest cumulative frequency percentage on a number basis are 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 0.8 μm or more, 1 μm or more, 1.2 μm or more, 1.4 μm or more, 1.5 μm or more, 1.7 μm or more, 1.9 μm or more, 2 μm or more, 4 μm or more, 6 μm or more, 8 μm or more, 1 μm or more, 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, 10 μm or more, 11 μm or more, 12 μm or more, 13 μm or more, 14 μm or more, 15 μm or more, 16 μm or more, 17 μm or more, 18 μm or more, 19 μm or more, 20 μm or more, 21 μm or more, 22 μm or more, 23 μm or more, 24 μm or more, 25 μm or more, 26 μm or more, 27 μm or more, 28 μm or more, 29 μm or more, 30 μm or more, 31 μm or more, 32 μm or more, 33 μm or more, 34 μm or more, 35 μm or more, 36 μm or more, 37 μm or more, 38 μm or more, 39 μm or more, 39 μm or more, 38 μm or more, 3 The thickness may be 0 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and may be 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, 2.5 μm or less, 2.3 μm or less, 2.1 μm or less, 1.9 μm or less, 1.7 μm or less, 1.6 μm or less, 1.4 μm or less, or 1.1 μm or less. For component a, the particle size (median diameter) that reaches 50% when calculated from the smallest cumulative frequency rate on a number basis is 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 1 μm or more, 1.5 μm or more, 1.8 μm or more, 2 μm or more, 2.2 μm or more, 2.4 μm or more, 2.5 μm or more, 2.7 μm or more, 2.9 μm or more, 4 μm or more, 6 μm or more, 8 μm or more, 10 μm or more, 15 μm or more, The thickness may be 20 μm or more, 25 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and may be 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, 4.5 μm or less, 4 μm or less, 3.5 μm or less, 3.2 μm or less, 3.0 μm or less, 2.8 μm or less, 2.7 μm or less, or 2.6 μm or less.For component a, the particle sizes that reach 90% when calculated from the smallest to the largest cumulative frequency percentage on a number basis are 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 0.8 μm or more, 1 μm or more, 1.2 μm or more, 1.4 μm or more, 1.5 μm or more, 1.7 μm or more, 1.9 μm or more, 2 μm or more, 3 μm or more, 3.5 μm or more, 3.8 μm or more, 4 μm or more, 4.2 μm or more, 4.4 μm or more, 4.5 μm or more, 4.7 μm or more, 4.9 μm or more, 5.3 μm or more, 5.5 μm or more, 6 μm or more, 7 μm or more, 10 μm or more, 15 μm or more , 20 μm or more, 25 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, or 300 μm or more, or 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, 8 μm or less, 7.5 μm or less, 7 μm or less, 6.5 μm or less, 6.2 μm or less, 6 μm or less, 5.8 μm or less, or 5.5 μm or less. These particle sizes are measured by the method described in the examples below.

[0043] In component a, the particle size that reaches 10% when calculated from the smallest cumulative frequency percentage on a number basis may be 0.1 to 50 μm, 0.3 to 40 μm, 0.5 to 30 μm, 0.7 to 10 μm, 0.9 to 5 μm, or 1 to 2 μm.

[0044] Component a may be particles having a large specific surface area. Component a, which is a particle having a large specific surface area, may be a cluster-like secondary particle formed by association of primary particles, or may be a porous particle. In this case, the amount of extracellular vesicles adsorbed can be further increased, and the sedimentation rate by centrifugation can be further improved.

[0045] When component a is a cluster-like secondary particle formed by association of primary particles, the particle size of the primary particles is, in average diameter, 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, 0.02 μm or more, 0.04 μm or more, 0.06 μm or more, 0.08 μm or more, 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 0.6 μm or more, 0.7 μm or more, 0.8 μm or more. The thickness may be 0.9 μm or more, 0.9 μm or more, or 1 μm or more, and may be 2 μm or less, 1.5 μm or less, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, 0.3 μm or less, 0.2 μm or less, 0.1 μm or less, 0.09 μm or less, 0.07 μm or less, 0.05 μm or less, 0.03 μm or less, or 0.01 μm or less.

[0046] The ratio of the average diameter of secondary particles to the average diameter of primary particles (average diameter of secondary particles / average diameter of primary particles) may be 1 or more, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 200 or more, 400 or more, 600 or more, 800 or more, 1000 or more, and may be 10,000 or less, 8,000 or less, 6,000 or less, 4,000 or less, 2,000 or less, 1,000 or less, 900 or less, 700 or less, 500 or less, 300 or less, 100 or less, 90 or less, 70 or less, 50 or less, 30 or less, 10 or less, 9 or less, 7 or less, 5 or less, 3 or less, or 2 or less. The average diameter of primary particles and the average diameter of secondary particles are measured by the following method. The average diameter of primary particles can be calculated by averaging the diameters measured along the major axes of 10 randomly selected particles in a microscopic image taken with a scanning electron microscope. The average diameter of the secondary particles can be measured by dynamic light scattering.

[0047] When component a is a porous particle, the inner diameter of the micropores possessed by the particle may be in the same range as the average diameter of the primary particles described above, and the ratio of the average diameter of the particle to the inner diameter of the micropores possessed by the particle may be in the same range as the ratio of the average diameter of the secondary particles to the average diameter of the primary particles described above.

[0048] When component a is separated from the liquid by centrifugation or the like, the interface between component a and the supernatant liquid is easily observed, which makes it easier to prevent the operational error of erroneously discarding component a and improves workability. Therefore, the total light transmittance of the liquid in which component a is dispersed may be 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. When observing extracellular vesicles adsorbed to component a using a phase-contrast microscope or a fluorescence microscope, the extracellular vesicles can be easily observed, and therefore the total light transmittance of the liquid in which component a is dispersed may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more. The total light transmittance of the liquid in which component a is dispersed may be 5 to 90%, 10 to 90%, 20 to 90%, 30 to 80%, 40 to 70%, or 50 to 60%.

[0049] The haze of the liquid in which component a is dispersed may be 0.5% or more, 1% or more, 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The haze of the liquid in which component a is dispersed may be 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The haze of the liquid in which component a is dispersed may be 0.5 to 100%, 10 to 100%, 30 to 100%, 50 to 100%, 70 to 100%, 90 to 100%, or 95 to 100%.

[0050] The total light transmittance and haze can be measured by the following method. A dried sample of component a is dispersed in ion-exchanged water, allowed to stand for 1 hour, and then centrifuged at 5000 g for 5 minutes to separate into a clear supernatant (upper layer) and a cloudy sediment (lower layer), and the supernatant is completely removed. Subsequently, ion-exchanged water is added to the sediment in an amount 14 times its volume. Ultrasonic waves are irradiated for 1 minute to disperse the precipitate. This liquid is placed in a cell with an optical path length of 1 cm, and using the cell containing ion-exchanged water as a reference, the total light transmittance is measured in accordance with JIS K 7375:2008, and the haze is measured in accordance with JIS K 7316:2000.

[0051] Since component a is suitable for improving the long-term storage stability of component a, it may have a peak in the 2θ range of 30° to 35° in the X-ray diffraction (XRD) spectrum. The peak in the 2θ range of 30° to 35° in the XRD spectrum of component a may be the peak with the highest intensity. Since component a is suitable for further improving the long-term storage stability of component a, the position of the peak in the 2θ range of 30° to 35° in the XRD spectrum may be 30° or more, 30.5° or more, 31° or more, 31.5° or more, 32° or more, or 35° or less, 33.5° or less, 32.5° or less, or 32° or less.

[0052] Component a may have a peak in the 2θ range of 28° or more and less than 30° in its X-ray diffraction (XRD) spectrum. The peak in the 2θ range of 28° or more and less than 30° (or 28.0° or more and less than 30.0°) may be the peak with the highest intensity in the XRD spectrum of component a. The position of the peak in the 2θ range of 28° or more and less than 30° in the XRD spectrum may be 28.5° or more and 30.0° or less, 29.0° or more and 30.0° or less, or 29.0° or more and 29.5° or less.

[0053] Component a may have a peak in the 2θ range of 10° to 15° in an X-ray diffraction (XRD) spectrum. The peak in the 2θ range of 10° to 15° may be the peak with the highest intensity in the XRD spectrum of component a. The position of the peak in the 2θ range of 10° to 15° in the XRD spectrum may be 10° to 13°, 10° to 12°, or 11° to 12°.

[0054] Component a is suitable for further increasing the solubility of component a in an aqueous chelating agent solution and facilitating desorption of extracellular vesicles adsorbed to component a, thereby further increasing the recovery rate of extracellular vesicles, and therefore the half width (full width at half maximum (FWHM)) of the most intense peak in the XRD spectrum may be in the range of 0.2° to 5.5°. The half width is the peak width at the position where the intensity of the most intense peak is half in the region where the most intense peak exists in the XRD spectrum. The half width of the most intense peak in the XRD spectrum was 0.2° or more, 0.5° or more, 0.7° or more, 0.9° or more, 1.0° or more, 1.1° or more, 1.2° or more, 1.3° or more, 1.4° or more, 1.5° or more, 1.6° or more, 1.7° or more, 1.8° or more, 1.9° or more, 2.0° or more, 2.5° or more, 3.0° or more, 4.0° or more, or 5.0° or more. and may be 5.5° or less, 4.5° or less, 3.5° or less, 3.0° or less, 2.5° or less, 2.2° or less, 2.1° or less, 2.0° or less, 1.9° or less, 1.8° or less, 1.7° or less, 1.6° or less, 1.5° or less, 1.4° or less, 1.3° or less, 1.2° or less, 1.1° or less, 1.0° or less, 0.8° or less, 0.6° or less, 0.4° or less, or 0.2° or less.

[0055] The content of components insoluble in the aqueous chelating agent solution is further reduced, and the amount of impurities remaining in the recovered extracellular vesicles is further reduced. Therefore, in the XRD spectrum of component a, the intensity of the peak at a 2θ value between 30° and 35° (PI １ ) with respect to the intensity of the peak (PI) at 2θ values ​​between 28° and less than 30° ２ ) ratio (PI ２ / PI １) may be 0.001 or more, 0.01 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.7 or more, 0.9 or more, 1.0 or more, 1.1 or more, 1.3 or more, 1.5 or more, 1.7 or more, or 1.9 or more, and may be 10.0 or less, 8.0 or less, 6.0 or less, 4.0 or less, 2.0 or less, 1.0 or less, 0.8 or less, 0.6 or less, 0.4 or less, 0.2 or less, 0.1 or less, 0.08 or less, 0.06 or less, 0.04 or less, 0.02 or less, or 0.01 or less. ２ / PI １ may be 0.001 to 10, 0.01 to 1, 0.01 to 0.5, 0.01 to 0.2, or 0.1 to 0.2.

[0056] Since it is suitable for further reducing the solubility in pure water and further increasing the adsorption rate of extracellular vesicles, the intensity of the peak (PI) in the 2θ value between 30° and less than 35° in the XRD spectrum is １ ) with respect to the intensity of the peak (PI) between 10° and 15° in 2θ value. ３ ) ratio (PI ３ / PI １ ) may be 0.001 or more, 0.01 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.7 or more, 0.9 or more, 1.0 or more, 1.1 or more, 1.3 or more, 1.5 or more, 1.7 or more, or 1.9 or more, and may be 10.0 or less, 8.0 or less, 6.0 or less, 4.0 or less, 2.0 or less, 1.0 or less, 0.8 or less, 0.6 or less, 0.4 or less, 0.2 or less, 0.1 or less, 0.08 or less, 0.06 or less, 0.04 or less, 0.02 or less, or 0.01 or less. ３ / PI １ may be 0.001 to 10, 0.001 to 1, 0.001 to 0.1, 0.001 to 0.05, or 0.01 to 0.05.

[0057] The XRD measurement is performed using CuKα radiation. The detailed conditions for the XRD measurement are as described in the Examples below.

[0058] Since this is more suitable for purifying extracellular vesicles using a sample containing extracellular vesicles and a chelating agent (such as EDTA plasma), the amount of chelating agent required to dissolve component a may be 0.1 mmol / g or more, 0.5 mmol / g or more, 1 mmol / g or more, 2 mmol / g or more, 3 mmol / g or more, 4 mmol / g or more, 5 mmol / g or more, 6 mmol / g or more, 7 mmol / g or more, 8 mmol / g or more, 9 mmol / g or more, 10 mmol / g or more, 11 mmol / g or more, 12 mmol / g or more, 13 mmol / g or more, 14 mmol / g or more, 15 mmol / g or more, 20 mmol / g or more, 50 mmol / g or more, 100 mmol / g or more, or 1000 mmol / g or more. Furthermore, since extracellular vesicles can be eluted with a low concentration or a small amount of chelating agent and the influence of the chelating agent on downstream analysis and testing can be further suppressed, the amount of chelating agent required to dissolve component a may be 1000 mmol / g or less, 500 mmol / g or less, 200 mmol / g or less, 150 mmol / g or less, 80 mmol / g or less, 60 mmol / g or less, 40 mmol / g or less, 20 mmol / g or less, 19 mmol / g or less, 18 mmol / g or less, 17 mmol / g or less, 16 mmol / g or less, 15 mmol / g or less, 14 mmol / g or less, 13 mmol / g or less, 12 mmol / g or less, 11 mmol / g or less, 10 mmol / g or less, 9 mmol / g or less, 8 mmol / g or less, 7 mmol / g or less, 6 mmol / g or less, 5 mmol / g or less, 4 mmol / g or less, 3 mmol / g or less, 2 mmol / g or less, or 1 mmol / g or less. The amount of chelating agent required to dissolve component a may be, for example, 0.1 to 1000 mmol / g, 1 to 100 mmol / g, 2 to 20 mmol / g, 5 to 10 mmol / g, or 7 to 9 mmol / g.

[0059] By treating extracellular vesicles with a small volume of component a, the amount of desorption solution added during elution can be further reduced, and the concentration rate of extracellular vesicles can be further improved. Therefore, the saturated adsorption amount (ng) of extracellular vesicles per unit weight (mg) of component a is a value based on the amount of microRNA contained in the extracellular vesicles, and can be 1 ng / mg or more, 10 ng / mg or more, 30 ng / mg or more, 50 ng / mg or more, 70 ng / mg or more, 90 ng / mg or more, 100 ng / mg or more, 200 ng / mg or more, 300 ng / mg or more, 400 ng / mg or more, 50 ng / mg or more, 60 ng / mg or more, 70 ng / mg or more, 90 ng / mg or more, 100 ng / mg or more, 200 ng / mg or more, 300 ng / mg or more, 400 ng / mg or more, 50 ng / mg or more, 60 ng / mg or more, 70 ng / mg or more, 8 ...50 ng / mg or more, 60 ng / mg or more, 70 ng / mg or more The concentration may be 00ng / mg or more, 600ng / mg or more, 700ng / mg or more, 800ng / mg or more, 900ng / mg or more, 1000ng / mg or more, 1100ng / mg or more, 1300ng / mg or more, 1500ng / mg or more, 1700ng / mg or more, 1900ng / mg or more, 2000ng / mg or more, 3000ng / mg or more, 4000ng / mg or more, 5000ng / mg or more, 7000ng / mg or more, 9000ng / mg or more, 10000ng / mg or more, or 100000ng / mg or more. Furthermore, when manually isolating extracellular vesicles from a culture medium in which cells producing a small amount of extracellular vesicles have been cultured, the amount of solid phase carrier added can be reduced while conditions can be set that make the vesicles easily visible. Therefore, the saturated adsorption amount (ng) of extracellular vesicles per unit weight (mg) of component a is a value based on the amount of microRNA contained in the extracellular vesicles, and can be set to 500,000 ng / mg or less, 50,000 ng / mg or less, 20,000 ng / mg or less, 10,000 ng / mg or less, 8,000 ng / mg or less, 6,000 ng / mg or less, 5,000 ng / mg or less, 4,500 ng / mg or less, 3,500 ng / mg or less, 25 The saturation level may be 00ng / mg or less, 2000ng / mg or less, 1800ng / mg or less, 1600ng / mg or less, 1400ng / mg or less, 1200ng / mg or less, 1000ng / mg or less, 950ng / mg or less, 850ng / mg or less, 750ng / mg or less, 650ng / mg or less, 550ng / mg or less, 450ng / mg or less, 350ng / mg or less, 250ng / mg or less, 150ng / mg or less, 100ng / mg or less, 80ng / mg or less, 60ng / mg or less, 40ng / mg or less, 20ng / mg or less, 10ng / mg or less, or 5ng / mg or less.The saturated adsorption amount (ng) of extracellular vesicles per unit weight (mg) of component a is a value based on the amount of microRNA contained in the extracellular vesicles, and may be 1 to 50,000 ng / mg, 10 to 10,000 ng / mg, 100 to 5,000 ng / mg, 500 to 2,000 ng / mg, or 1,000 to 1,500 ng / mg.

[0060] The saturated adsorption amount of extracellular vesicles can be calculated from the dry weight (mg) of component a and the amount of microRNA contained in the adsorbed extracellular vesicles using the following formula when component a is added under conditions such that the amount of extracellular vesicles does not become 10% or less of the original amount by adding component a: Amount of microRNA adsorbed to solid phase carrier / Dry weight of added solid phase carrier [ng / mg]

[0061] Component a is a phosphate ion (PO ４ ３－ ), calcium ions (Ca ２＋ ) and carbonate ions (CO ３ ２－ Component a may contain at least one ion selected from the group consisting of phosphate ions (PO ４ ３－ ) as a constituent ion, and at least phosphate ion (PO ４ ３－ ) and calcium ions (Ca ２＋ ) as a constituent ion. The carbonate ion in this specification is a carbonate ion (H ２ CO ３ ) includes all ions that can be generated by the dissociation of the carbonate ion, HCO ３ － and CO ３ ２－ The phosphate ion in this specification refers to phosphate (H ３ P.O. ４ ) includes all ions that can be generated by dissociation of the phosphate ion. ２ P.O. ４ － , H.P.O. ４ ２－ , and P.O. ４ ３－ It may exist in either state.

[0062] Phosphate ions (PO ４ ３－ ) to calcium ions (Ca ２＋ The number of moles of phosphate ions (PO ) in component a may be the same as the range of moles of calcium element per mole of phosphorus element in component a described above. ４ ３－ ) to carbonate ion (CO ３ ２－ The number of moles of carbon element per mole of phosphorus element in component a) may be the same as the numerical range of the number of moles of carbon element per mole of phosphorus element in component a described above.

[0063] Component a may be a reaction product of a carbonate, a phosphate, and a calcium salt (hereinafter referred to as "reactant a1"). Specifically, component a (material for extracellular vesicle purification) can be obtained by a method including the step of reacting a carbonate, a phosphate, and a calcium salt in an aqueous solution to obtain component a1 (material for extracellular vesicle purification) as a white precipitate.

[0064] Carbonates are CO ３ ２－ or HCO ３ － and its counter ion. ３ ２－ or HCO ３ － The counter ion may be at least one selected from the group consisting of alkali metal ions (e.g., lithium ions, sodium ions, and potassium ions), alkaline earth metal ions (e.g., magnesium ions, calcium ions, strontium ions, and barium ions), and ammonium ions. The carbonate may be at least one selected from the group consisting of sodium bicarbonate, calcium carbonate, magnesium carbonate, potassium carbonate, lithium carbonate, ammonium carbonate, strontium carbonate, and barium carbonate. The carbonate may be sodium bicarbonate, as this is suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing loss during washing, and further increasing the recovery rate of extracellular vesicles.

[0065] Phosphate is PO ４ ３－, H.P.O. ４ ２－ or H ２ P.O. ４－ and its counter ion. ４ ３－ , H.P.O. ４ ２－ or H ２ P.O. ４－ The counter ion may be, for example, at least one selected from the group consisting of alkali metal ions and alkaline earth metal ions. The phosphate may be, for example, at least one selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, calcium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium hydrogen phosphate, ammonium hydrogen phosphate, strontium hydrogen phosphate, barium hydrogen phosphate, trisodium phosphate, and tripotassium phosphate. The phosphate may be 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, which is suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing loss during washing, and further increasing the recovery rate of extracellular vesicles.

[0066] Calcium salts are Ca ２＋ It is a compound consisting of Ca and its counter ion. ２＋ The counter ion may be, for example, at least one selected from the group consisting of inorganic acid ions (e.g., halide ions, nitrate ions, sulfate ions) and organic acid ions (e.g., acetate ions). The calcium salt may be, for example, at least one selected from the group consisting of calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium sulfate, calcium acetate, and calcium carbonate. The calcium salt may be calcium chloride or calcium carbonate, as these are suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing loss during washing, and further increasing the recovery rate of extracellular vesicles.

[0067] Component a may be a reaction product of a phosphate and a calcium salt (hereinafter referred to as "reactant a2"). Specifically, component a (material for extracellular vesicle purification) can also be obtained by a method including a step of reacting a phosphate with a calcium salt in an aqueous solution to obtain reactant a2 (material for extracellular vesicle purification) as a white precipitate.

[0068] Reactant a may be a reaction product of at least one carbonate selected from the group consisting of ammonium carbonate, magnesium carbonate, and sodium hydrogen carbonate, at least one phosphate selected from the group consisting of dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, and at least one calcium salt selected from the group consisting of calcium carbonate and calcium chloride.Reactant a may be a reaction product of at least one phosphate selected from the group consisting of dipotassium hydrogen phosphate, trisodium phosphate, and disodium hydrogen phosphate, and at least one calcium salt selected from the group consisting of calcium carbonate and calcium chloride.

[0069] Examples of methods for obtaining reactant a1 include a method of simultaneously mixing a carbonate, a phosphate, and a calcium salt to cause a reaction between these salts, and a method of mixing two salts selected from a carbonate, a phosphate, and a calcium salt to obtain a reaction mixture, and then mixing the remaining salt to obtain reactant a1. Examples of methods for obtaining reactant a2 include a method of mixing a phosphate and a calcium salt to cause a reaction between them to obtain reactant a2.

[0070] The reaction to obtain the above-described reactant a is carried out in a reaction solution obtained by mixing raw materials with water. In this specification, examples of water include distilled water, pure water, and ultrapure water (such as Milli-Q water). Milli-Q water refers to ultrapure water obtained using a Milli-Q water production system manufactured by Merck Millipore (Merck). The raw material may be an ion source capable of forming at least one ion selected from the group consisting of carbonate ions, phosphate ions, and calcium ions upon contact with water. The ion source for carbonate ions may be a carbonate salt. The ion source for phosphate ions may be a phosphate salt. The ion source for calcium ions may be a calcium salt. Calcium carbonate, which is a source of carbonate ions and calcium ions, may also be used as an ion source. The reaction solution may contain an acid (e.g., nitric acid) as needed.

[0071] The concentration of carbonate in the reaction solution may be 50 mg / L or more, 100 mg / L or more, 200 mg / L or more, 300 mg / L or more, 400 mg / L or more, 500 mg / L or more, 600 mg / L or more, 700 mg / L or more, 800 mg / L or more, 900 mg / L or more, 1000 mg / L or more, 1500 mg / L or more, 2000 mg / L or more, 2500 mg / L or more, 3000 mg / L or more, 3500 mg / L or more, 4000 mg / L or more, 4500 mg / L or more, 5000 mg / L or more, 5500 mg / L or more, 6000 mg / L or more, 6500 mg / L or more, 7000 mg / L or more, or 7500 mg / L or more, based on the total volume of the reaction solution, because this makes it easier to obtain component a with a superior recovery rate of extracellular vesicles. The concentration of carbonate in the reaction solution may be 9000 mg / L or less, 8000 mg / L or less, 7000 mg / L or less, 6000 mg / L or less, 5000 mg / L or less, 4000 mg / L or less, 3000 mg / L or less, 2000 mg / L or less, 1000 mg / L or less, 500 mg / L or less, 400 mg / L or less, 300 mg / L or less, 200 mg / L or less, or 150 mg / L or less, based on the total volume of the reaction solution, since this makes it easier to obtain component a with a superior recovery rate of extracellular vesicles. The concentration of carbonate in the reaction solution may be 50 to 9000 mg / L, 100 to 5000 mg / L, 500 to 3000 mg / L, or 1500 to 2500 mg / L, based on the total volume of the reaction solution.

[0072] The concentration of phosphate in the reaction solution may be 90 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, 1500 mg / L or more, 2000 mg / L or more, 2500 mg / L or more, 2600 mg / L or more, or 3000 mg / L or more, based on the total volume of the reaction solution, since this makes it easier to obtain component a with a superior recovery rate of extracellular vesicles. The concentration of phosphate in the reaction solution may be 100 g / L or less, 90 g / L or less, 60 g / L or less, 40 g / L or less, 20 g / L or less, 10 g / L or less, 8000 mg / L or less, 4000 mg / L or less, 3500 mg / L or less, 3000 mg / L or less, 2000 mg / L or less, 1500 mg / L or less, 1000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, 200 mg / L or less, 150 mg / L or less, or 120 mg / L or less, based on the total volume of the reaction solution, since this makes it easier to obtain component a which has a better recovery rate of extracellular vesicles. The concentration of phosphate in the reaction solution may be 90 to 100,000 mg / L, 200 to 10,000 mg / L, 500 to 5,000 mg / L, or 500 to 2,000 mg / L based on the total volume of the reaction solution.

[0073] The concentration of calcium salt in the reaction solution may be 20 mg / L or more, 40 mg / L or more, 100 mg / L or more, 160 mg / L or more, 180 mg / L or more, 200 mg / L or more, 300 mg / L or more, 400 mg / L or more, 500 mg / L or more, 600 mg / L or more, 700 mg / L or more, 800 mg / L or more, 900 mg / L or more, 1000 mg / L or more, 1100 mg / L or more, 1200 mg / L or more, 1300 mg / L or more, 1400 mg / L or more, or 1500 mg / L or more, based on the total volume of the reaction solution, because this makes it easier to obtain component a with a superior recovery rate of extracellular vesicles. The concentration of calcium salt in the reaction solution may be 30 g / L or less, 20 g / L or less, 10 g / L or less, 8000 mg / L or less, 5000 mg / L or less, 3000 mg / L or less, 1800 mg / L or less, 1500 mg / L or less, 1000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, 300 mg / L or less, 250 mg / L or less, 200 mg / L or less, or 150 mg / L or less, based on the total volume of the reaction solution, since this makes it easier to obtain component a with a superior recovery rate of extracellular vesicles. The concentration of calcium salt in the reaction solution may be 20 to 30,000 mg / L, 50 to 10,000 mg / L, 100 to 1,000 mg / L, or 150 to 250 mg / L, based on the total volume of the reaction solution.

[0074] The extracellular vesicle purification kit may include a solid phase carrier containing component a at least on its surface. The configuration of the solid phase carrier is not particularly limited other than the surface component. The solid phase carrier may be a solid phase carrier containing a core portion and a surface layer containing component a. The solid phase carrier may be a solid phase carrier whose core portion and surface layer are composed only of component a, or may be a solid phase carrier containing a core portion containing a component other than component a and a surface layer containing component a.

[0075] As the solid phase carrier whose core and surface layer are composed only of component a, the above-mentioned reactants a1 and a2 can be used as they are.

[0076] A solid phase carrier comprising a core portion containing a component other than component a and a surface layer containing component a can be obtained by a method including a step of coating the surface of the core portion with component a. The method for coating the surface of the core portion with component a is not particularly limited and can be appropriately selected depending on the type and shape of the core portion, etc. Methods for coating the core portion with component a include sputtering, chemical vapor deposition, dip coating, spray coating, electrostatic adsorption, and embedding by particle collision in a high-velocity airflow.

[0077] Examples of components other than component a include a component that is soluble in both pure water and an aqueous chelating agent solution, a component that is insoluble in both pure water and an aqueous chelating agent solution, and a component that is soluble in pure water but insoluble in an aqueous chelating agent solution.

[0078] The shape of the solid phase carrier can be appropriately selected depending on the application, such as particles, pellets, powder, granules, fibers, membranes, substrates, tubes, bags, rings, plates, etc. The solid phase carrier may be packed into a column for use.

[0079] The solid phase carrier may be a magnetically responsive material. When the solid phase carrier contains a magnetically responsive solid phase carrier, the solid phase carrier can be separated using a magnet, making it easier to separate the solid phase carrier from the liquid.

[0080] The solid phase carrier may further include an intermediate layer between the core portion and the surface layer. Figure 1 shows one embodiment of a solid phase carrier including a core portion, an intermediate layer, and a surface layer in this order. The solid phase carrier 10 shown in Figure 1 includes a core portion 1, an intermediate layer 6 that covers at least a portion of the surface of the core portion 1, and a surface layer 5 that covers at least a portion of the surface of the intermediate layer 6.

[0081] (Core portion) The shape of the core portion is not limited to particles, and pellets, powders, granules, fibers, films, substrates, tubes, bags, rings, plates, etc. can be appropriately selected. The core portion may have irregularities with a large specific surface area, such as a porous shape or a shape with protrusions. Since this is suitable for coating the surface of the core portion 1 with even more minute magnetic bodies, the core portion 1 may have irregularities with a large specific surface area, such as a porous shape or a shape with protrusions.

[0082] Examples of the shape of the particulate core portion 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 better magnetic responsiveness are easily obtained, the particle size of the core portion may be 0.1 μm or more, 0.5 μm or more, 1.0 μm or more, 1.5 μm or more, or 2.0 μm or more. Since magnetic particles with better redispersibility in aqueous solvents are easily obtained, the particle size of the core portion may be 100 μm or less, 80 μm or less, 60 μm or less, 40 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, or 3 μm or less. The particle size of the core portion may be 0.1 μm or more to 100 μm or less, 0.5 μm or more to 10 μm or less, 1.0 μm or more to 5.0 μm or less, or 2.0 μm or more to 3.0 μm or less. The particle size of the core portion can be calculated by determining the mode diameter from the particle size distribution measured by dynamic light scattering.

[0083] The constituent component of the core part may be a polymer or a metal oxide. When the core part is composed of a polymer, the specific gravity of the solid phase carrier can be reduced compared to when the main component of the core part is a component with a high specific gravity (a magnetic material or a metal oxide), and the dispersibility of the solid phase carrier in a solvent is further improved. Improved dispersibility makes it easier to obtain a particulate solid phase carrier that can be uniformly dispersed with little stirring force. A particulate solid phase carrier that can be uniformly dispersed can improve the reproducibility of extracellular vesicle purification. The metal oxide as a constituent component of the core part 1 may be a material exemplified as the metal oxide in the surface layer described below.

[0084] 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 unit.

[0085] The styrene-based monomer may be, for example, at least one selected from the group consisting of 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.

[0086] Examples of the (meth)acrylate monomer 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; acrylate and other alkoxy(cyclo)alkyl(meth)acrylates; polyvalent (meth)acrylates such as trimethylolpropane tri(meth)acrylate; cyanoacrylates such as 2-cyanoethyl(meth)acrylate, 2-cyanopropyl(meth)acrylate, and 3-cyanopropyl(meth)acrylate; hydroxymethyl(meth)acrylate, 2-hydroxyethyl(meth)acrylate, 6-hydroxyhexyl(meth)acrylate, and 4-hydroxycyclohexyl(meth)acrylate; The (meth)acrylate may be at least one selected from the group consisting of substituted hydroxy(meth)acrylates such as glycidyl (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.

[0087] The vinyl ester monomer may be, for example, at least one selected from the group consisting of vinyl acetate, vinyl propionate, and vinyl versatate.

[0088] (Intermediate Layer) The intermediate layer may be a metal oxide layer containing a metal oxide, or may be an extracellular vesicle non-adhesive layer. The metal oxide can be appropriately selected depending on the type of extracellular vesicles of interest and the type of sample containing extracellular vesicles to be purified. The metal oxide may be at least one selected from the group consisting of 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.

[0089] The intermediate layer may be an extracellular vesicle non-adhesive layer, since this further improves the recovery rate of extracellular vesicles. When the intermediate layer is an extracellular vesicle non-adhesive layer, the extracellular vesicles are detached and the extracellular vesicle non-contact layer is exposed on the surface of the solid phase carrier due to dissolution of the surface layer containing component a upon contact with the chelating agent. The exposure of the extracellular vesicle non-contact layer inhibits readsorption of the detached extracellular vesicles to the surface of the solid phase carrier. As a result, it is believed that the recovery rate of extracellular vesicles can be further improved.

[0090] Examples of materials constituting the non-adhesive layer of extracellular vesicles include surfactants, proteins, and polymers. The surfactant may be a surfactant having a polyethylene glycol structure. Cationic, anionic, or nonionic surfactants can be used. Examples of proteins that can be used include bovine serum albumin, skim milk, and serum.

[0091] The polymer may be a polymer containing at least one monomer unit selected from the group consisting of a styrene-based monomer unit and a (meth)acrylate-based monomer unit. Specific examples of the styrene-based monomer unit and the (meth)acrylic acid monomer unit are as described above. The (meth)acrylate-based monomer unit may be, for example, at least one selected from the group consisting of hydroxymethyl methacrylate, hydroxyethyl methacrylate, and 2-methacryloyloxyethyl phosphorylcholine.

[0092] The content of the styrene-based monomer unit may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, or 70 parts by mass or more, and 90 parts by mass or less, 80 parts by mass or less, 60 parts by mass or less, or 40 parts by mass or less, relative to 100 parts by mass of the total amount of the monomer units constituting the polymer. The content of the styrene-based monomer unit may be 10 to 90 parts by mass, 10 to 80 parts by mass, 10 to 60 parts by mass, 10 to 40 parts by mass, 30 to 90 parts by mass, 30 to 80 parts by mass, 30 to 60 parts by mass, 30 to 40 parts by mass, 50 to 90 parts by mass, 50 to 80 parts by mass, 50 to 60 parts by mass, 70 to 90 parts by mass, or 70 to 80 parts by mass, relative to 100 parts by mass of the total amount of the monomer units constituting the polymer.

[0093] 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, or 70 parts by mass or more, and may be 90 parts by mass or less, 80 parts by mass or less, 60 parts by mass or less, or 40 parts by mass or less, relative to 100 parts by mass of the total amount of monomer units constituting the polymer. The content of the (meth)acrylate monomer units may be 10 to 90 parts by mass, 10 to 80 parts by mass, 10 to 60 parts by mass, 10 to 40 parts by mass, 30 to 90 parts by mass, 30 to 80 parts by mass, 30 to 60 parts by mass, 30 to 40 parts by mass, 50 to 90 parts by mass, 50 to 80 parts by mass, 50 to 60 parts by mass, 70 to 90 parts by mass, or 70 to 80 parts by mass, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0094] The content of the (meth)acrylic acid monomer unit may be 5 parts by mass or more, 10 parts by mass or more, or 20 parts by mass or more, and may be 40 parts by mass or less, 30 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of all the monomer units constituting the polymer. The content of the (meth)acrylic acid monomer unit may be 5 to 40 parts by mass, 5 to 30 parts by mass, 5 to 15 parts by mass, 10 to 40 parts by mass, 10 to 30 parts by mass, 10 to 15 parts by mass, 20 to 40 parts by mass, or 20 to 30 parts by mass, relative to 100 parts by mass of all the monomer units constituting the polymer.

[0095] The content of the (meth)acrylic acid ester monomer unit may be 10 parts by mass or more, 30 parts by mass or more, 50 parts by mass or more, or 70 parts by mass or more, and may be 90 parts by mass or less, 80 parts by mass or less, 60 parts by mass or less, or 40 parts by mass or less, relative to 100 parts by mass of all the monomer units constituting the polymer. The content of the (meth)acrylic acid ester monomer unit may be 10 to 90 parts by mass, 10 to 80 parts by mass, 10 to 60 parts by mass, 10 to 40 parts by mass, 30 to 90 parts by mass, 30 to 80 parts by mass, 30 to 60 parts by mass, 30 to 40 parts by mass, 50 to 90 parts by mass, 50 to 80 parts by mass, 50 to 60 parts by mass, 70 to 90 parts by mass, or 70 to 80 parts by mass, relative to 100 parts by mass of all the monomer units constituting the polymer.

[0096] Examples of methods for forming an intermediate layer containing a polymer on the surface of the core include a method in which a polymer is formed on the surface of the core by a polymerization reaction of a monomer in a reaction solution containing the material constituting the core, 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.

[0097] (Micromagnetic Material) The solid phase carrier may contain a micromagnetic material. The micromagnetic material is a particle having magnetic responsiveness and smaller than the core portion. The particle size of the micromagnetic material 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. The micromagnetic material may be superparamagnetic. The particle size of the micromagnetic material may be 0.001 to 1 μm, 0.001 to 0.5 μm, 0.001 to 0.1 μm, or 0.001 to 0.05 μm. The micromagnetic material may be a nano-sized magnetic material (nanomagnetic material).

[0098] The particle size of the minute magnetic particles is 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.

[0099] The material of the minute magnetic body can be, for example, iron oxide such as magnetite.

[0100] The surface of the minute magnetic particles may contain a cationic dispersant or anionic dispersant.

[0101] The micromagnetic material may be a commercially available product, such as EMG607 (trade name, manufactured by Ferrotec Corporation).

[0102] The micromagnetic particles may be dispersed in the intermediate layer, or may cover the core portion between the core portion and the intermediate layer and be coated with the intermediate layer. By dispersing the micromagnetic particles in the intermediate layer or coating the surface of the micromagnetic particles with the intermediate layer, the acid resistance of the solid phase carrier can be further improved.

[0103] Since the magnetic response is further improved, the content of the micromagnetic particles per 1 g of the solid phase carrier may be 0.1 g / g or more, 0.2 g / g or more, 0.5 g / g or more, or 0.7 g / g or more. Since the dispersibility of the solid phase carrier in an aqueous solvent is further improved, the content of the micromagnetic particles per 1 g of the solid phase carrier may be 0.9 g / g or less, 0.8 g / g or less, 0.7 g / g or less, or 0.6 g / g or less. The content of the magnetic particles per 1 g of the solid phase carrier may be, for example, 0.1 to 0.9 g / g, 0.1 to 0.8 g / g, 0.1 to 0.7 g / g, 0.1 to 0.6 g / g, 0.2 to 0.9 g / g, 0.2 to 0.8 g / g, 0.2 to 0.7 g / g, 0.2 to 0.6 g / g, 0.5 to 0.9 g / g, 0.5 to 0.8 g / g, 0.5 to 0.7 g / g, 0.5 to 0.6 g / g, 0.7 to 0.9 g / g, or 0.7 to 0.8 g / g.

[0104] From the viewpoint of controlling the specific gravity of the solid phase carrier and facilitating adjustment of the sedimentation rate of the solid phase carrier, the weight proportion of the micromagnetic material contained in the solid phase carrier may be 5 to 70 mass%, 10 to 60 mass%, 15 to 50 mass%, or 20 to 40 mass% based on the total mass of the solid phase carrier. The content of the micromagnetic material is measured by ICP (Inductively Coupled Plasma) emission spectrometry.

[0105] A solid phase carrier containing minute magnetic particles can be obtained by a method including a step of incorporating minute magnetic particles into the solid phase carrier (magnetization step) at any timing during the production process of the solid phase carrier.

[0106] The magnetization step may be a step of contacting the core part with the minute magnetic substance to adsorb (physical adsorb) the minute magnetic substance to the surface of the core part. The method of contacting the core part with the minute magnetic substance is not particularly limited, but the magnetization step can be carried out by stirring in a liquid phase or a gas phase.

[0107] In the magnetization process, the amount of micromagnetic material 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. The amount of the minute magnetic material used relative to 1 g of the core portion is, for example, 0.5 to 2.0 g, 0.5 to 1.9 g, 0.5 to 1.8 g, 0.5 to 1.7 g, 0.5 to 1.6 g, 0.5 to 1.5 g, 0.6 to 2.0 g, 0.6 to 1.9 g, 0.6 to 1.8 g, 0.6 to 1.7 g, 0.6 to 1.6 g, 0.6 to 1.5 g, 0.7 to 2.0 g, 0.7 to 1.9 g, 0.7 to 1.8 g, 0.7 to 1.7 g, 0.7 to 1.6 g, 0.7 to 1.5 g, 0.8 to 2.0 g, 0.8 to 1.9 g,

[0033] The amount of the hydroxybenzoate may be 0.8 to 1.8g, 0.8 to 1.7g, 0.8 to 1.6g, 0.8 to 1.5g, 0.9 to 2.0g, 0.9 to 1.9g, 0.9 to 1.8g, 0.9 to 1.7g, 0.9 to 1.6g, 0.9 to 1.5g, 1.0 to 2.0g, 1.0 to 1.9g, 1.0 to 1.8g, 1.0 to 1.7g, 1.0 to 1.6g, 1.0 to 1.5g, 1.1 to 2.0g, 1.1 to 1.9g, 1.1 to 1.8g, 1.1 to 1.7g, 1.1 to 1.6g, or 1.1 to 1.5g.

[0108] Examples of methods for adsorbing the micromagnetic substance to the surface of the core part include a method in which the core part is brought into contact with a micromagnetic substance having a surface charge opposite to that of the core part, thereby spontaneously adsorbing the micromagnetic substance to the surface of the core part through electrostatic interaction, a method in which the micromagnetic substance is bonded to the surface of the core part through a chemical reaction between a functional group on the surface of the core part and the micromagnetic substance, a method in which the core part and the micromagnetic substance are combined by applying mechanical energy, a method in which a hydrophobic micromagnetic substance is brought into contact with a hydrophobic core part, and a method in which a micromagnetic substance is brought into contact with a paramagnetic core part. The method for adsorbing the micromagnetic substance to the surface of the core part may be a combination of several methods. For example, the micromagnetic substance may be spontaneously adsorbed to the surface of the core part through electrostatic interaction, dried, and then further mechanical energy may be applied to firmly adsorb the micromagnetic substance to the core part.

[0109] The method of spontaneously adsorbing the micromagnetic particles to the surface of the core part by electrostatic interaction is suitable for further increasing the amount of micromagnetic particles adsorbed to the core part, and therefore may be a method of mixing the core part and the micromagnetic particles in an aqueous solution. When the core part and the micromagnetic particles are mixed in an aqueous solution, the aqueous solution may contain an electrolyte such as sodium chloride. When the aqueous solution contains an electrolyte, the electrolyte concentration in the aqueous solution may be a concentration at which the micromagnetic particles do not aggregate with each other, and may be, for example, a concentration of about 0.01 to 0.5 M.

[0110] The reaction conditions (reaction temperature and reaction time) when adsorbing the micromagnetic particles to the surface of the core part by electrostatic interaction can be set appropriately depending on the type of material used, etc. The reaction temperature when adsorbing the micromagnetic particles to the surface of the core part 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 micromagnetic particles to the surface of the core part 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. The reaction time for adsorbing the nano-magnetic particles onto the surface of the core portion may be, for example, 1.0 to 5.0 hours, 1.0 to 4.0 hours, 1.0 to 3.0 hours, 1.5 to 5.0 hours, 1.5 to 4.0 hours, 1.5 to 3.0 hours, 2.0 to 5.0 hours, 2.0 to 4.0 hours, 2.0 to 3.0 hours, 3.0 to 5.0 hours, 3.0 to 4.0 hours, or 4.0 to 5.0 hours.

[0111] The method of compounding the core portion and the minute magnetic material by applying mechanical energy may be a method capable of processing materials in a dry manner using an apparatus for compounding by swirling force in a high-speed air stream, an apparatus for mixing and stirring using a rotation-revolution mixer or a ball mill, an apparatus for surface coating using a spray dryer, etc. Examples of apparatus for compounding by applying mechanical energy include the Hybridization System NHS Series (manufactured by Nara Machinery Works, Ltd.), Nobilta NOB (manufactured by Hosokawa Micron Corporation), High-Speed ​​Agitation Type Powder Spheroidizer NSM Series (manufactured by Seishin Enterprise Co., Ltd.), and Mini Spray Dryer B-290 Model (Shibata Scientific Co., Ltd.).

[0112] When compounding is performed by the swirling force in a high-speed airflow, the rotation speed is set to 8000 min, which is suitable for fixing the minute magnetic particles more firmly to the core part. －１ That's all, 9000 min －１ That's all, 10,000 min －１ or more, or 11,000 min －１ The rotation speed is preferably 15,000 min or more, since this is suitable for forming the particles into a spherical shape. －１ Below, 14000min －１ Below, 13000min－１ or less, or 12,000 min －１ The rotation speed may be, for example, 8000 to 15000 min －１ ,8000~14000min －１ ,8000~13000min －１ ,8000~12000min －１ ,9000~15000min －１ ,9000~14000min －１ ,9000~13000min －１ ,9000~12000min －１ , 10000~15000min －１ , 10000~14000min －１ , 10000~13000min －１ , 10000~12000min －１ , 11000~15000min －１ , 11000~14000min －１ , 11000~13000min －１ , or 11,000 to 12,000 min －１ The treatment time may be 1 to 60 minutes, 1 to 20 minutes, 1 to 10 minutes, or 3 to 10 minutes, as this is suitable for forming the particles into a spherical shape. The treatment temperature may be 25 to 80°C, 25 to 60°C, 25 to 50°C, or 25 to 40°C, as this is suitable for suppressing fusion of the particles.

[0113] Since this is suitable for uniformly adsorbing the minute magnetic particles to the core portion, the minute magnetic particles may be electrostatically adsorbed to the core portion in an aqueous solution.

[0114] In order to more firmly fix the adsorbed micromagnetic particles to the surface of the core portion, the material after the composite in the aqueous solution may be dried. The 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, or 80°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, or 55°C or lower. The drying temperature may be, for example, 40 to 80°C, 40 to 70°C, 40 to 65°C, 40 to 60°C, 40 to 55°C, 45 to 80°C, 45 to 70°C, 45 to 65°C, 45 to 60°C, 45 to 55°C, 50 to 80°C, 50 to 70°C, 50 to 65°C, 50 to 60°C, 50 to 55°C, 55 to 80°C, 55 to 70°C, 55 to 65°C, 55 to 60°C, 60 to 80°C, 60 to 70°C, or 60 to 65°C. The drying time may be, for example, 0.5 hours or more, 1 hour or more, 3 hours or more, 5 hours or more, or 10 hours or more, and may be 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less. The drying time may be, for example, 0.5 to 20 hours, 0.5 to 15 hours, 0.5 to 10 hours, 0.5 to 5 hours, 1 to 20 hours, 1 to 15 hours, 1 to 10 hours, 1 to 5 hours, 3 to 20 hours, 3 to 15 hours, 3 to 10 hours, 3 to 5 hours, 5 to 20 hours, 5 to 15 hours, 5 to 10 hours, 10 to 20 hours, or 10 to 15 hours.

[0115] The core parts to which the micro-magnetic particles are adsorbed may be dispersed in a high-speed airflow after drying. By dispersing the core parts to which the micro-magnetic particles are adsorbed in a high-speed airflow, the aggregates of the core parts can be dissociated by the impact force. While dispersing the core parts to which the micro-magnetic particles are adsorbed in a high-speed airflow, the core parts can be surface-modified in a dry manner by the impact force between the core parts, thereby making the shape of the core parts spherical. The high-speed airflow treatment can be carried out using the above-mentioned device that performs compounding by swirling force in a high-speed airflow.

[0116] (Surface Treatment) The surfaces of the solid phase carrier and the fine magnetic particles may be treated with an inorganic surface modifier such as a silane coupling agent, if necessary. The silane coupling agent may be, for example, 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 at least one selected from the group consisting of 3-isocyanatepropyltriethoxysilane. The surface of the minute magnetic particles may contain a cationic dispersant or anionic dispersant.

[0117] [Second embodiment] The kit for extracellular vesicle purification according to the second embodiment includes 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)");

[0118] [Component (A)] The shape of the solid phase carrier can be appropriately selected 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 may be a magnetically responsive material. When a magnetically responsive solid phase carrier is included, the solid phase carrier can be separated using a magnet, making it easier to separate the solid phase carrier from the liquid.

[0119] 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.

[0120] The solid phase carrier may be 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.

[0121] 2 and 3 are schematic diagrams (cross-sectional views) showing examples of solid phase carriers. The solid phase carriers will be described in more detail below using as an example a particulate solid phase carrier having a metal oxide on its surface as shown in FIGS.

[0122] The solid support 10 shown in FIG. 2 has a metal oxide 3 on its surface. In the solid support 10, the metal oxide 3 is present on the surface of the 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. 2. Since the solid support 10 is a particulate solid support containing micromagnetic particles 2, it is a magnetic particle having the metal oxide 3 on its surface. The micromagnetic particles 2 may be dispersed in the surface layer 4 as shown in FIG. 2. FIG. 3 is a schematic diagram (cross-sectional view) showing another example of a solid support. As shown in FIG. 3, the micromagnetic particles 2 may be present between the metal oxide 3 and the core portion 1.

[0123] <Surface Layer> The surface layer 4 contains a metal oxide 3. The surface layer 4 may further contain minute magnetic bodies 2 as required.

[0124] (Metal Oxide) 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.

[0125] 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.

[0126] Since the metal oxide 3 is more likely to adsorb extracellular vesicles, it may be 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 extracellular vesicles can be further improved.

[0127] From the viewpoint of making it easier to desorb extracellular vesicles at a pH near neutral, where denaturation of extracellular vesicles is unlikely to occur, the metal oxide 3 may be at least one selected from the group consisting of silica, indium oxide, and indium tin oxide.

[0128] From the viewpoint of further 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, the metal oxide 3 may be at least one selected from the group consisting of tin oxide, indium oxide, and indium tin oxide.

[0129] From the viewpoint of making it easier to remove phenol red contained in the culture medium and to recover extracellular vesicles with higher purity, the metal oxide 3 may be at least one selected from the group consisting of tin oxide, indium oxide, and indium tin oxide.

[0130] From the viewpoint of making it easier to adsorb extracellular vesicles in blood or a sample containing serum or plasma, the metal oxide 3 may be at least one selected from the group consisting of silica and titanium oxide.

[0131] From the viewpoint of further increasing acid resistance and making it easier to recover extracellular vesicles with a higher content of microRNA, the metal oxide 3 may be silica.

[0132] From the viewpoint of making it easier to recover extracellular vesicles with a high content of tetraspanins such as CD63, the metal oxide 3 may be at least one selected from the group consisting of indium oxide and indium tin oxide.

[0133] From the viewpoint of making it easier to collect extracellular vesicles with a high protein content, the metal oxide 3 may be titanium oxide.

[0134] 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 may be bound to the metal oxide 3 present on the surface of the solid support by electrostatic interaction, hydrophobic interaction, hydrogen bonding, coordinate bonding, etc. From the viewpoint of stable dispersibility, various functional groups of the compound may form bonds such as coordinate bonds with the metal oxide 3 present on the surface of the solid support.

[0135] By binding an anionic functional group to the metal oxide 3 present on the surface of the solid phase carrier, the surface of the solid phase carrier 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 phase carrier. Furthermore, the method facilitates detachment of extracellular vesicles from the solid phase carrier, further improving the detachment rate. The anionic functional group may be a carboxy group, a sulfo group, or a phosphate group. Methods for binding an anionic functional group to the metal oxide 3 include modifying the metal oxide 3 with a compound such as 3-trimethoxysilylpropyl succinic acid, 3-trimethoxysilylpropyl sulfonate, or 3-trimethoxysilylpropyl phosphonate, and reacting the hydroxyl group of the metal oxide 3 with succinic anhydride to introduce a carboxy group.

[0136] 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. The cationic functional group may be an amino group (unsubstituted amino group or substituted amino group), a guanidinium group, a pyridinium group, or an imidazolium group. Methods for binding the cationic functional group to the metal oxide 3 include methods of 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.

[0137] 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. The hydrophobic functional group may be an alkyl group having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, or a butyl group, a phenyl group, an alkenyl group, or an alkynyl group. Methods for bonding the hydrophobic functional group to the surface of the solid support include methods of modifying the metal oxide 3 with methyltrimethoxysilane, methyltriethoxysilane, hexyltrimethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, octyltrimethoxysilane, octyltriethoxysilane, isobutyltrimethoxysilane, hexadecyltrimethoxysilane, dimethyldiethoxysilane, diphenyldimethoxysilane, or the like.

[0138] By binding 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 bound 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 the metal oxide 3 include methoxypolyethylene glycol, polyethylene glycol distearate, polyethylene glycol monomethyl ether, and polyethylene glycol sorbitan monooleate.

[0139] For the purpose of controlling the specific gravity of the solid phase carrier 10 and adjusting the sedimentation rate of the solid phase carrier 10, the content of the metal oxide 3 contained in the solid phase carrier 10 may be 0.1 to 10 mass%, 0.5 to 5 mass%, 1 to 5 mass%, or 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.

[0140] (Micromagnetic body) The micromagnetic body 2 is a particle having magnetic response and smaller than the core part. The particle size, material, commercially available micromagnetic body, etc. of the micromagnetic body 2 may be the same as those described in the first embodiment.

[0141] The surface of the magnetic particles 2 may contain lipids and inorganic surface modifiers such as a silane coupling agent. The silane coupling agent may be the same as that described in the first embodiment. The surface of the magnetic particles 2 may contain a cationic dispersant or anionic dispersant.

[0142] The micromagnetic particles 2 may be dispersed in the surface layer 4, or may be disposed between the core portion 1 and the layer made of metal oxide 3, covering the core portion and also being covered by the metal oxide 3. When the micromagnetic particles 2 are dispersed in the surface layer 4, or when the surface of the micromagnetic particles 2 is covered with the metal oxide 3, the acid resistance of the solid phase carrier 10 can be improved.

[0143] The content of the micro magnetic particles 2 per gram of the solid phase carrier and the weight ratio of the micro magnetic particles 2 contained in the solid phase carrier 10 may be as described in the first embodiment.

[0144] When the 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.

[0145] (Thickness of Surface Layer) 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 may be 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, or 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 may be 0.1 μm or less, 0.05 μm or less, 0.02 μm or less, or 0.01 μm or less. The thickness of the surface layer 4 can be calculated by measuring the thickness of the surface layer 4 for 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.

[0146] <Core Portion> The shape, particle size, constituent components, and other aspects of the core portion 1 may be as described above.

[0147] The core portion 1 may contain minute magnetic particles as needed. As the minute magnetic particles that can be contained in the surface layer 4, those exemplified above can be used.

[0148] 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.

[0149] When a paramagnetic covering layer is provided, the thickness of the paramagnetic covering layer may be 0.001 to 1 μm, 0.001 to 0.1 μm, 0.001 to 0.01 μm, or 0.001 to 0.005 μm, since the residual magnetization of the core part 1 is further reduced and aggregation of the core parts 1 is further suppressed. The thickness of the paramagnetic covering layer can be calculated by measuring the thickness of the layer for 10 or more core parts 1 in an image of the cross section of the core part 1 observed with a transmission electron microscope and calculating the average value.

[0150] 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 on 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.

[0151] (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 5 m ２ / g or more, 10m ２ / g or more, or 20m ２ The specific surface area of ​​the solid support may be 100 m / g or more because the mechanical strength of the solid support can be further improved. ２ / g or less, 90m ２ / g or less, 80m ２ / g or less, 70m ２ / g or less, 60m ２ / g or less, 50m ２ / g or less, 40m ２ / g or less, or 30m ２ The specific surface area of ​​the solid phase carrier 10 may be, for example, 2 to 100 m ２ / g, 2-90m ２ / g, 2-80m ２ / g, 2-70m ２ / g, 2-60m ２ / g, 2-50m ２ / g, 2-40m ２ / g, 2-30m ２ / g, 5-100m ２ / g, 5-90m ２ / g, 5-80m ２ / g, 5-70m ２ / g, 5-60m ２ / g, 5-50m２ / g, 5-40m ２ / g, 5-30m ２ / g, 10-100m ２ / g, 10-90m ２ / g, 10-80m ２ / g, 10-70m ２ / g, 10-60m ２ / g, 10-50m ２ / g, 10-40m ２ / g, 10-30m ２ / g, 20-100m ２ / g, 20-90m ２ / g, 20-80m ２ / g, 20-70m ２ / g, 20-60m ２ / g, 20-50m ２ / g, or 20 to 40 m ２ / g, 20-30m 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.

[0152] <Method for producing a solid support having a metal oxide on its surface> The solid support 10 having a metal oxide on its surface can be obtained, for example, by a method including a step of coating the core part 1 with a metal oxide 3 (coating step).

[0153] When the solid phase carrier 10 contains 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. Details of the magnetization step may be as described above.

[0154] (Coating Step) In the coating step, the core part 1 is coated with a metal oxide 3. The method for coating the core part 1 with the metal oxide 3 is not particularly limited, but examples thereof include a method in which the core part 1 having the micromagnetic material 2 adsorbed thereon is dispersed in a solution containing a reagent and a reaction catalyst that are raw materials for the metal oxide 3, and the metal oxide 3 is formed on the surface of the core part 1 by allowing the reaction to proceed, a method in which the core part 1 having the micromagnetic material 2 adsorbed thereon and powder of the metal oxide 3 are dispersed in a high-speed air current to form the metal oxide 3 on the surface of the core part 1, and a method in which the surface of the core part 1 is coated with a metal oxide or a raw material thereof.

[0155] The method for coating the core 1 with the metal oxide 3 may be a sol-gel method in which the core 1 having the nano-magnetic bodies 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 the metal oxide 3 added during the sol-gel reaction is more suitable for forming a sufficient amount of metal oxide 3 on the surface of the core portion 1, and may be 0.05 g or more, 0.1 g or more, 0.2 g or more, or 0.5 g or more per 1 g of the core portion. The amount of the precursor of the metal oxide 3 added during the sol-gel reaction is more suitable for suppressing aggregation of the core portion 1 during the reaction, and may be 5 g or less, 2 g or less, 1 g or less, or 0.5 g or less per 1 g of the core portion. The amount of the reaction catalyst added during the reaction is more suitable for forming a sufficient amount of metal oxide on the surface of the core portion 1, and may be 20 mL or more, 30 mL or more, 40 mL or more, or 50 mL or more per 1 L of solvent. The amount of the reaction catalyst added during the reaction is more suitable for suppressing aggregation of the core portion 1 during the reaction, and may be 100 mL or less, 80 mL or less, 70 mL or less, or 60 mL or less per 1 L of solvent.

[0156] 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. As the metal salt, a salt of a metal ion and its counterion can be used. 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.

[0157] The reaction solvent used in forming the metal oxide 3 may be ethanol, 2-propanol, or butanol, or may be ethanol or 2-propanol, or may be 2-propanol, since these solvents are more suitable for uniformly coating the metal oxide 3 while suppressing aggregation of the core portion 1.

[0158] 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 may be 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.

[0159] <Solid support having a polymer on its surface> The solid support may be a solid support having 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 a styrene-based monomer unit and a (meth)acrylate-based monomer unit. Specific examples of the styrene-based monomer unit and the (meth)acrylic acid monomer unit 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.

[0160] 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.

[0161] 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, and may be polyoxyethylene alkyl ethers having a long-chain alkyl having 1 to 20 carbon atoms.

[0162] The molecular weight of the polyethylene glycol or polyoxyethylene may be 500 or more, 1000 or more, 1500 or more, or 2000 or more. The molecular weight of the polyethylene glycol or polyoxyethylene may be 20000 or less, 10000 or less, 5000 or less, or 3000 or less. The molecular weight of the polyethylene glycol or polyoxyethylene may be 500 to 20,000, 500 to 10,000, 500 to 5,000, 500 to 3,000, 1,000 to 20,000, 1,000 to 10,000, 1,000 to 5,000, 1,000 to 3,000, 1,500 to 20,000, 1,500 to 10,000, 1,500 to 5,000, 1,500 to 3,000, 2,000 to 20,000, 2,000 to 10,000, 2,000 to 5,000, or 2,000 to 3,000. The molecular weight is measured by the following method. Using gel permeation chromatography (GPC), a sample of polyethylene glycol or polyoxyethylene is dissolved in an appropriate solvent, such as tetrahydrofuran (THF) or water, and a calibration curve is prepared using polyethylene glycols of known molecular weights as standard samples. The relative molecular weight (number average molecular weight Mn) is calculated by comparing the retention time of the sample with that of the standard samples. A refractive index detector or a light scattering detector can be used as the detector.

[0163] 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.

[0164] The content of the styrene-based monomer unit 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 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 the monomer units constituting the polymer. The content of the styrene-based monomer unit may be 10 to 95 parts by mass, 10 to 75 parts by mass, 10 to 55 parts by mass, 10 to 35 parts by mass, 10 to 15 parts by mass, 30 to 95 parts by mass, 30 to 75 parts by mass, 30 to 55 parts by mass, 30 to 35 parts by mass, 50 to 95 parts by mass, 50 to 75 parts by mass, 50 to 55 parts by mass, 70 to 95 parts by mass, 70 to 75 parts by mass, or 90 to 95 parts by mass, relative to 100 parts by mass of the total amount of the monomer units constituting the polymer.

[0165] 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. The content of the (meth)acrylate monomer units may be 10 to 95 parts by mass, 10 to 75 parts by mass, 10 to 55 parts by mass, 10 to 35 parts by mass, 10 to 15 parts by mass, 30 to 95 parts by mass, 30 to 75 parts by mass, 30 to 55 parts by mass, 30 to 35 parts by mass, 50 to 95 parts by mass, 50 to 75 parts by mass, 50 to 55 parts by mass, 70 to 95 parts by mass, 70 to 75 parts by mass, or 90 to 95 parts by mass, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0166] 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 all the monomer units constituting the polymer. The content of the (meth)acrylic acid monomer unit may be 1 to 50 parts by mass, 1 to 30 parts by mass, 1 to 20 parts by mass, 1 to 10 parts by mass, 5 to 50 parts by mass, 5 to 30 parts by mass, 5 to 20 parts by mass, 5 to 10 parts by mass, 10 to 50 parts by mass, 10 to 30 parts by mass, 10 to 20 parts by mass, 20 to 50 parts by mass, 20 to 30 parts by mass, or 40 to 50 parts by mass, relative to 100 parts by mass of all the monomer units constituting the polymer.

[0167] 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. The content of the (meth)acrylic acid ester monomer units may be 10 to 95 parts by mass, 10 to 75 parts by mass, 10 to 55 parts by mass, 10 to 35 parts by mass, 10 to 15 parts by mass, 30 to 95 parts by mass, 30 to 75 parts by mass, 30 to 55 parts by mass, 30 to 35 parts by mass, 50 to 95 parts by mass, 50 to 75 parts by mass, 50 to 55 parts by mass, 70 to 95 parts by mass, 70 to 75 parts by mass, or 90 to 95 parts by mass, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0168] 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. The monomer or surfactant having a polyethylene glycol structure or a polyoxyethylene structure may be 10 to 95 parts by mass, 10 to 75 parts by mass, 10 to 55 parts by mass, 10 to 35 parts by mass, 10 to 15 parts by mass, 30 to 95 parts by mass, 30 to 75 parts by mass, 30 to 55 parts by mass, 30 to 35 parts by mass, 50 to 95 parts by mass, 50 to 75 parts by mass, 50 to 55 parts by mass, 70 to 95 parts by mass, 70 to 75 parts by mass, or 90 to 95 parts by mass, relative to 100 parts by mass of the total amount of monomer units constituting the polymer.

[0169] 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 the polymer on its surface may be treated with a silane coupling agent as needed. Specific examples of silane coupling agents are as described above.

[0170] [Component (B)] 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) contains carbonate ions (CO ３ ２－ ), phosphate ions (PO ４ 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 a compound or mixture containing two or more constituent ions selected from the group consisting of H ２ CO ３ , HCO３ － and CO ３ ２－ In this specification, the phosphate ion that can be contained as component (B) includes phosphoric acid and ions generated by dissociation of phosphoric acid. In component (B), the phosphate ion can be present in any of the following states: H ３ P.O. ４ , H ２ P.O. ４ － , H.P.O. ４ ２－ , and P.O. ４ ３－ It may exist in either state.

[0171] 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 composite inorganic salts thereof).

[0172] 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.

[0173] 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 solid phase carriers to which extracellular vesicles have been 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 carrier to which extracellular vesicles have been 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, but 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 carrier by contacting the solid phase carrier with a liquid that does not contain the two or more ions.

[0174] 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.

[0175] 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) may be a component containing a carbonate, a phosphate, and a calcium salt, since this further improves the adsorption rate and recovery rate of extracellular vesicles. The carbonate, phosphate, and calcium salt are as described above.

[0176] The carbonate may be sodium bicarbonate, as this is suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing losses during washing, and further increasing the recovery rate of extracellular vesicles from the solid phase carrier.

[0177] The phosphate may be 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, or may be sodium dihydrogen phosphate, as this is suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing loss during washing, and further increasing the recovery rate of extracellular vesicles from the solid phase carrier.

[0178] The calcium salt may be calcium chloride, which is suitable for further increasing the adsorption rate of extracellular vesicles, further suppressing losses during washing, and further increasing the recovery rate of extracellular vesicles from the solid phase carrier.

[0179] The content of carbonate per 1 mol of phosphate may be 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. The content of carbonate per 1 mol of phosphate may be, for example, 50 mol or more, or 100 mol or more.

[0180] The content of carbonate per 1 mol of phosphate may be 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 adsorption of extracellular vesicles to the solid phase carrier and further improve the recovery rate of extracellular vesicles. The content of carbonate per 1 mol of phosphate 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.

[0181] The content of carbonate per 1 mol of phosphate can further promote the adsorption of extracellular vesicles to the solid phase carrier and can further improve the recovery rate of extracellular vesicles, and is, for example, 0.1 to 200 mol, 0.1 to 150 mol, 0.1 to 80 mol, 0.1 to 55 mol, 0.5 to 200 mol, 0.5 to 150 mol, 0.5 to 80 mol, 0.5 to 55 mol, 1 to 200 mol, 1 to 150 mol 1, 1 to 80 mol, 1 to 55 mol, 5 to 200 mol, 5 to 150 mol, 5 to 80 mol, 5 to 55 mol, 10 to 200 mol, 10 to 150 mol, 10 to 80 mol, 10 to 55 mol, 30 to 200 mol, 30 to 150 mol, 30 to 80 mol, 30 to 55 mol, 45 to 200 mol, 45 to 150 mol, 45 to 80 mol, or 45 to 55 mol.

[0182] 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.

[0183] The content of carbonate per 1 mol of calcium salt may be 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 adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles. The content of carbonate per 1 mol of calcium salt may be, for example, 25 mol or more, or 50 mol or more.

[0184] The content of carbonate per 1 mol of calcium salt may be 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. The content of carbonate per 1 mol of calcium salt 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.

[0185] The content of carbonate per 1 mol of calcium salt can further promote the adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles, and is therefore preferably 0.05 to 150 mol, 0.05 to 80 mol, 0.05 to 55 mol, 0.05 to 40 mol, 0.05 to 30 mol, 0.3 to 150 mol, 0.3 to 80 mol, 0.3 to 55 mol, 0.3 to 40 mol, 0.3 to 30 mol, 1 to 15 0 mol, 1 to 80 mol, 1 to 55 mol, 1 to 40 mol, 1 to 30 mol, 6 to 150 mol, 6 to 80 mol, 6 to 55 mol, 6 to 40 mol, 6 to 30 mol, 10 to 150 mol, 10 to 80 mol, 10 to 55 mol, 10 to 40 mol, 10 to 30 mol, 20 to 150 mol, 20 to 80 mol, 20 to 55 mol, 20 to 40 mol, or 20 to 30 mol.

[0186] 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.

[0187] The content of calcium salt per 1 mol of phosphate can be 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, because this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles. The content of calcium salt per 1 mol of phosphate can be, for example, 2 mol or more, 3 mol or more, or 5 mol or more.

[0188] The content of calcium salt per 1 mol of phosphate can be 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, because this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles. The content of calcium salt per 1 mol of phosphate can be, for example, 1 mol or less, 0.7 mol or less, 0.4 mol or less, or 0.2 mol or less.

[0189] The content of calcium salt per 1 mol of phosphate may be 0.1 to 20 mol, 0.1 to 10 mol, 0.1 to 6 mol, 0.1 to 4 mol, 0.1 to 2.5 mol, 0.5 to 20 mol, 0.5 to 10 mol, 0.5 to 6 mol, 0.5 to 4 mol, 0.5 to 2.5 mol, 1 to 20 mol, 1 to 10 mol, 1 to 6 mol, 1 to 4 mol, 1 to 2.5 mol, 1.6 to 20 mol, 1.6 to 10 mol, 1.6 to 6 mol, 1.6 to 4 mol, or 1.6 to 2.5 mol, because this can further promote adsorption of extracellular vesicles and further improve the recovery rate of extracellular vesicles.

[0190] The content (mol) of calcium ions per 1 mol of phosphate ions may be in the same numerical range as the "content of calcium salt per 1 mol of phosphate" described above.

[0191] The total content of the (B) component (and the total content of carbonates, phosphates, and calcium salts) 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 per 1 g of the solid phase carrier, or 2 mmol or more, 3 mmol or more, or 4 mmol or more. The total content of the (B) component (and the total content of carbonates, phosphates, and calcium salts) may be 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, or 8 mmol or less, 7 mmol or less, or 6 mmol or less per 1 g of the solid phase carrier.

[0192] The total content of component (B) (and the total content of carbonate, phosphate, and calcium salt) may be 0.01 to 2000 mmol, 0.1 to 1000 mmol, 1 to 100 mmol, or 1 to 10 mmol per 1 g of the solid phase carrier.

[0193] 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.

[0194] 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 for purifying extracellular vesicles.

[0195] In the extracellular vesicle purification kit according to the second embodiment, the component (B) may be mixed with the component (A), or may not be mixed with the component (A).

[0196] <Chelating Agent> The extracellular vesicle purification kit may further contain a chelating agent. 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, the chelating agent can also be referred to as a desorption promoter. In this specification, the term "chelating agent" refers to a compound (multidentate ligand) that has multiple coordination sites within the molecule and is multidentate with respect to metal ions, and the term "chelating action" refers to multidentate coordination (binding) with respect to metal ions.

[0197] The chelating agent may be at least one selected from the group consisting of polycarboxylic acid compounds, polyphosphate compounds, polyphenol compounds, other compounds having chelating activity, and salts thereof.

[0198] The polycarboxylic acid compound is a compound having multiple carboxy groups. The polycarboxylic acid compound may be at least one selected from the group consisting of aminopolycarboxylic acids having at least one amino group and multiple carboxy groups, and polycarboxylic acids having no amino groups but multiple carboxy groups (non-aminopolycarboxylic acids). The aminopolycarboxylic acid may be at least one selected from the group consisting of 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), and HEDTA (hydroxyethylethylenediaminetriacetic acid). The non-aminopolycarboxylic acid may be at least one selected from the group consisting of oxalic acid, polyacrylic acid, and citric acid.

[0199] The polyvalent phosphate compound is a compound having a plurality of phosphate groups. The polyvalent phosphate compound may be phytic acid. The polyhydric phenol compound is a compound having a plurality of phenolic hydroxy groups. The polyhydric phenol compound may be at least one selected from the group consisting of polyphenols and calixarenes. The other compound having chelating activity may be at least one selected from the group consisting of desferrioxamine and penicillamine.

[0200] The chelating agent may be a polycarboxylic acid compound or an aminopolycarboxylic acid, as these are more suitable for promoting detachment of extracellular vesicles from solid phase carriers.

[0201] The chelating agent may be in a solid state (powder, etc.) or in a solution state. A chelating agent in a solution state can be prepared by dissolving the chelating agent in a solvent such as water. A chelating agent in a solution state can be diluted as necessary when used for purifying extracellular vesicles.

[0202] When the extracellular vesicle purification kit includes a solid phase carrier, the total content of the chelating agent may be 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 the solid phase carrier. To further promote the detachment of extracellular vesicles and suppress the denaturation of extracellular vesicles, the total content of the chelating agent may be 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 the solid phase carrier. The total content of the chelating agent may be, for example, 0.001 to 10 μmol, 0.1 to 5 μmol, or 1 to 2 μmol per 1 g of the solid phase carrier.

[0203] <Other Components> The extracellular vesicle purification kit may further contain other components in addition to the components described above. Examples of other components include a pH adjuster, a protein remover, and the like. The protein remover is used to remove proteins from a liquid containing extracellular vesicles as a pretreatment. There are no particular limitations on the type of protein remover. The protein remover may be, for example, at least one selected from the group consisting of ammonium sulfate, trichloroacetic acid, acetone, chloroform, methanol, phenol, and mixtures thereof. When the extracellular vesicle purification kit contains component (B), the component corresponding to (B) is excluded from the pH adjuster.

[0204] [Method for purifying extracellular vesicles] The method for purifying extracellular vesicles according to this embodiment is a method for purifying extracellular vesicles using the extracellular vesicle purification kit according to the first or second embodiment described above.

[0205] <Purification method according to the first embodiment> The purification method according to the first embodiment includes, for example, an adsorption step of contacting a liquid containing extracellular vesicles with component a to adsorb the extracellular vesicles to component a, a separation step of separating component a to which the extracellular vesicles have adsorbed from the liquid, and a desorption step of contacting component a to which the separated extracellular vesicles have adsorbed with a chelating agent to dissolve at least a portion of component a and detach the extracellular vesicles from component a.

[0206] The method for purifying extracellular vesicles according to the first embodiment may further include a pH adjustment step of adjusting the liquid containing the extracellular vesicles to an acidic pH (to make the liquid acidic) before the adsorption step. The method for purifying extracellular vesicles may further include a step of washing component a adsorbed by the extracellular vesicles with a washing solution after the separation step and before the desorption step.

[0207] Below, the method for purifying extracellular vesicles according to the first embodiment will be described in more detail using as an example a method including a pH adjustment step, an adsorption step, a separation step, a washing step, and a desorption step in this order.

[0208] <pH Adjustment Step> In the pH adjustment step, the pH of the liquid containing extracellular vesicles is adjusted to make it an acidic liquid.

[0209] Examples of liquids containing extracellular vesicles include culture media in which cells are cultured (culture supernatants), blood, serum, plasma, urine, sweat, saliva, and breast milk.

[0210] 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. The pH may be 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more, as this makes it easier to suppress the denaturation of extracellular vesicles. The pH may be less than 7, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, as this can further improve the adsorption rate of extracellular vesicles. The pH may be, for example, 0 or more and less than 7, 3 or more and less than 7, or 5 or more and less than 7.

[0211] 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.

[0212] <Adsorption step> In the adsorption step, a liquid containing extracellular vesicles is mixed with component a, and the extracellular vesicles are adsorbed onto the surface of component a. Since component a is a component that is insoluble in pure water, component a does not dissolve even when brought into contact with a liquid in which the extracellular vesicles are dispersed in an aqueous solution such as a culture medium, and is therefore able to adsorb the extracellular vesicles. Furthermore, since component a is a component that is insoluble in pure water, it becomes possible to wash the surface of component a to which the extracellular vesicles are adsorbed with an aqueous solution such as a buffer, thereby preventing impurities from being mixed into the extracellular vesicles that are finally recovered.

[0213] The adsorption step can be carried out by mixing a liquid containing extracellular vesicles with a solid phase carrier having the above-mentioned component a on at least its surface.

[0214] Contact between a liquid containing extracellular vesicles and component a can be achieved, for example, by adding a solid carrier having component a at least on its surface to a liquid containing extracellular vesicles, followed by stirring to disperse the solid 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 to ensure sufficient contact of the extracellular vesicles in the liquid containing extracellular vesicles with component a, or 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, or may be 30 minutes or less, 20 minutes or less, or 10 minutes or less to facilitate suppression of denaturation of the extracellular vesicles. The adsorption time may be, for example, 1 to 30 minutes, 1 to 15 minutes, 1 to 10 minutes, 3 to 30 minutes, 3 to 15 minutes, or 3 to 10 minutes.

[0215] In the adsorption step, to further increase the adsorption rate of extracellular vesicles, the amount of solid phase carrier added may be 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. Since it is easier to suppress the incorporation of impurities derived from the solid phase carrier, the amount of solid phase carrier added may be 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. The amount of solid phase carrier added may be 1 to 100 mg, 2 to 50 mg, or 5 to 10 mg per mL of liquid containing extracellular vesicles.

[0216] <Separation step> In the separation step, component a adsorbed by extracellular vesicles is separated from the liquid. Separation methods include centrifugation, filtration, natural sedimentation, etc. When magnetic particles are used, the separation method may be separation using a magnet.

[0217] <Washing Step> In the washing step, component a to which extracellular vesicles are adsorbed, obtained in the separation step, is washed. The liquid for washing component a (washing liquid) may be an aqueous solution that does not contain a chelating agent. Examples of the washing liquid include a buffer solution and an acidic or alkaline liquid. Examples of the buffer solution include acetate buffer, phosphate buffer, citrate buffer, Tris-HCl buffer, HEPES buffer, etc. The pH of the buffer solution used as the washing liquid may be 5.0 or more and 10.0 or less, 6.0 or more and 10.0 or less, greater than 7.0 and 10.0 or less, or 8.0 or more and 10.0 or less. The acidic or alkaline liquid may be a liquid within the above pH range.

[0218] The washing method is not particularly limited, and examples include a method in which component a is mixed with a washing solution and the washing solution is stirred. The temperature of the washing solution in the washing step may be 10 to 40°C or 15 to 25°C. The washing time in the washing step (e.g., the stirring time of the washing solution) can be appropriately selected depending on the type of washing solution, the temperature of the washing solution, and the like. The washing time may be, for example, 1 minute or more, or 3 minutes or more, and may be 30 minutes or less, 15 minutes or less, or 10 minutes or less. The washing time may be, for example, 1 to 30 minutes, 1 to 15 minutes, 1 to 10 minutes, 3 to 30 minutes, 3 to 15 minutes, or 3 to 10 minutes.

[0219] The separation step and the 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 the washing step, it is possible to remove a larger amount of residues other than extracellular vesicles adsorbed to component a.

[0220] <Desorption step> In the desorption step, component a to which the separated extracellular vesicles have adsorbed is contacted with a chelating agent to dissolve at least a portion of component a and desorb the extracellular vesicles from component a. Because component a is a component that dissolves in an aqueous chelating agent solution, at least a portion of component a is dissolved upon contact with the chelating agent, and the extracellular vesicles are desorbed from component a.

[0221] Methods for desorbing extracellular vesicles from component a include contacting component a to which extracellular vesicles have been adsorbed with a liquid for desorbing the extracellular vesicles (desorption liquid).

[0222] The desorption liquid may be a liquid containing a chelating agent, such as the chelating agents exemplified in the extracellular vesicle purification kit.

[0223] In order to promote desorption of extracellular vesicles, the content of the chelating agent in the desorption solution may be 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 amount of the desorption solution. The content of the chelating agent in the desorption solution may be 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, in order to promote desorption of extracellular vesicles and suppress denaturation of extracellular vesicles. The content of the chelating agent in the desorption solution may be 0.01 to 1000 mmol / L, 0.1 to 100 mmol / L, or 1 to 10 mmol / L, based on the total amount of the desorption solution.

[0224] The pH of the desorption liquid may be 6 or higher, and in order to further increase the desorption rate of extracellular vesicles, it may be 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 further suppress denaturation of extracellular vesicles, the pH of the liquid used in the desorption step may be 14 or lower, 13 or lower, 12 or lower, 11 or lower, 10 or lower, 9 or lower, or 8 or lower. The pH may be, for example, 7 or higher and 14 or lower, 7 or higher and 11 or lower, or 7 or higher and 9 or lower.

[0225] The desorption liquid may be an alkaline liquid, which makes it easier to desorb extracellular vesicles from the solid phase carrier.

[0226] 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.

[0227] 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 in order 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. In order to suppress denaturation of the extracellular vesicles, 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. The desorption time may be, for example, 1 to 30 minutes, 1 to 15 minutes, 1 to 10 minutes, 3 to 30 minutes, 3 to 15 minutes, or 3 to 10 minutes.

[0228] 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 component a, 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.

[0229] The method for purifying extracellular vesicles according to the first embodiment may be capable of adsorbing 50% or more, 60% or more, 70% or more, or 80% or more of the extracellular vesicles contained in a serum-free medium sample. The method for purifying extracellular vesicles according to the first embodiment may be capable of adsorbing 20% ​​or more, 40% or more, 60% or more, or 70% or more of the extracellular vesicles contained in a serum-containing sample.

[0230] The method for purifying extracellular vesicles may be capable of desorbing 10% or more, 30% or more, 50% or more, or 70% or more of the extracellular vesicles captured by the solid phase carrier for a serum-free medium sample.The method for purifying extracellular vesicles may be capable of desorbing 10% or more, 20% or more, 40% or more, or 60% or more of the extracellular vesicles captured by the solid phase carrier for a serum-containing sample.

[0231] <Purification method according to the second embodiment> The purification method according to the second embodiment includes an adsorption step of mixing a liquid containing extracellular vesicles with component (A) and component (B) and adsorbing the extracellular vesicles to the surface of component (A), a separation step of separating component (A) to which the extracellular vesicles have been adsorbed from the liquid, a washing step of washing component (A) to which the extracellular vesicles have been adsorbed, and a desorption step of detaching the extracellular vesicles from component (A) to which the extracellular vesicles have been adsorbed.

[0232] The method for purifying extracellular vesicles according to the second embodiment may further include a pH adjustment step of adjusting the liquid containing extracellular vesicles to an acidic pH (making it an acidic liquid) before the adsorption step.

[0233] Below, the extracellular vesicle purification method according to the second embodiment will be described in more detail using as an example a method including a pH adjustment step, an adsorption step, a separation step, a washing step, and a desorption step in this order.

[0234] <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 making the pH acidic (hydrogen ion exponent), the solid phase carrier and proteins tend to be positively charged, while the extracellular vesicles are negatively charged even in the acidic state, making it easier for the extracellular vesicles to be selectively adsorbed onto the surface of the solid phase carrier.

[0235] The liquid containing extracellular vesicles and the pH adjustment step, such as the pH adjustment method, may be as described in the purification method according to the first embodiment.

[0236] <Adsorption step> 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 step is carried out in the presence of component (B), which contains carbonate ions, phosphate ions, and calcium ions, thereby increasing the adsorption rate of extracellular vesicles onto the surface of the solid phase carrier.

[0237] The components (A) and (B) may be mixed in a solid state with a liquid containing extracellular vesicles, or an aqueous solution containing the components (A) and / or (B) may be prepared in advance and mixed in the aqueous solution state with a liquid containing extracellular vesicles.

[0238] 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, and 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, or 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, in order to facilitate suppression of denaturation of the extracellular vesicles, or may be 30 minutes or less, 20 minutes or less, or 10 minutes or less. The adsorption time may be, for example, 1 to 30 minutes, 1 to 15 minutes, 1 to 10 minutes, 3 to 30 minutes, 3 to 15 minutes, or 3 to 10 minutes.

[0239] The amount of solid phase carrier added in the adsorption step may be the same as that described in the purification method according to the first embodiment.

[0240] In order to further promote adsorption of extracellular vesicles, the total amount of carbonate, phosphate, and calcium salt added may be 1 mg / L or more, 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 relative to the total amount of the liquid containing the extracellular vesicles and the amount of the (B) component in aqueous solution used. Furthermore, in order to further facilitate desorption of the adsorbed extracellular vesicles, the total amount of carbonate, phosphate, and calcium salt added during adsorption may be 10,000 mg / L or less, 8,000 mg / L or less, 6,000 mg / L or less, 4,000 mg / L or less, 2,000 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, or 200 mg / L or less, relative to the total amount of the liquid containing the extracellular vesicles and the amount of the component (B) in the aqueous solution state. The total amount of carbonate, phosphate, and calcium salt added may be, for example, 1 to 10,000 mg / L, 10 to 5,000 mg / L, 100 to 2,000 mg / L, or 500 to 1,500 mg / L, relative to the total amount of the liquid containing the extracellular vesicles and the amount of the component (B) in the aqueous solution state.

[0241] The concentration of carbonate in the solution of component (B) when contacting the liquid containing extracellular vesicles with component (B) in the solution state is, based on the total amount of the liquid containing extracellular vesicles used and the amount of component (B) in the 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. The concentration of carbonate in the component (B) in a solution state when the liquid containing extracellular vesicles is brought into contact with the component (B) in a solution state may be 50 to 500,000 mg / L, 100 to 100,000 mg / L, 500 to 10,000 mg / L, or 1,000 to 5,000 mg / L, based on the total amount of the liquid containing extracellular vesicles used and the component (B) in an aqueous solution state used.

[0242] The concentration of phosphate in the component (B) in a solution state when contacting the liquid containing extracellular vesicles with the 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, or 100 mg / L or more, based on the total amount of the liquid containing extracellular vesicles used and the component (B) in an aqueous solution state used, and may be 200 mg / L or more. The concentration may be 1000 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, 2,000 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, 200 mg / L or less, 150 mg / L or less, or 120 mg / L or less. The concentration of phosphate in the component (B) in a solution state when the liquid containing extracellular vesicles is brought into contact with the component (B) in a solution state may be, for example, 1 to 20,000 mg / L, 10 to 10,000 mg / L, 100 to 5,000 mg / L, or 500 to 2,000 mg / L, based on the total amount of the liquid containing extracellular vesicles used and the component (B) in an aqueous solution state used.

[0243] The concentration of calcium salt in the (B) component in the state of solution 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 amount of the liquid containing extracellular vesicles used and the amount of the (B) component in the state of an aqueous solution used, and may be 30,000 mg / L or less, 2500 mg / L or less, The concentration may be 0 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. The concentration of the calcium salt in the component (B) in the form of a solution may be, for example, 1 to 30,000 mg / L, 10 to 10,000 mg / L, 50 to 5,000 mg / L, or 100 to 1,000 mg / L, based on the total amount of the liquid containing extracellular vesicles used and the amount of the component (B) in the form of an aqueous solution used.

[0244] <Separation step> In the separation step, the component (A) adsorbed by the extracellular vesicles is separated from the liquid. The separation method is not particularly limited, but separation using a magnet may be used for ease of operation. The separation method may be a method in which separation using a magnet is combined with centrifugation, filtration, natural sedimentation, etc. as necessary.

[0245] <Washing step> In the washing step, the component (A) to which extracellular vesicles are adsorbed, obtained in the separation step, is washed. Examples of liquids (washing liquids) for washing the component (A) include buffer solutions, component (B) in the form of an aqueous solution, and acidic or alkaline liquids. The buffer solutions and acidic or alkaline liquids may be as described in the purification method according to the first embodiment.

[0246] The washing method is not particularly limited, and examples thereof include a method in which the component (A) is mixed with a washing solution and the washing solution is stirred. The temperature of the washing solution and the washing time (e.g., the stirring time of the washing solution) in the washing step may be as described in the purification method according to the first embodiment.

[0247] The separation step and the washing step may be repeated in this order to repeatedly wash the component (A) to which the extracellular vesicles have been adsorbed with the washing solution. By repeatedly performing the separation step and the washing step, it is possible to remove a larger amount of residues other than the extracellular vesicles adsorbed to the component (A).

[0248] <Desorption step> In the desorption step, the extracellular vesicles are desorbed from the component (A) to which they have been adsorbed. Examples of methods for desorbing the extracellular vesicles from the component (A) include a method of contacting the component (A) to which the extracellular vesicles have been adsorbed with a liquid for desorbing the extracellular vesicles (desorption liquid).

[0249] In the purification method according to the second embodiment, the type of desorption liquid, the content of the chelating agent in the desorption liquid, the pH of the desorption liquid, the method for adjusting the pH of the desorption liquid, the desorption time, and other aspects may be the same as those described in the purification method according to the first embodiment.

[0250] 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 phase carrier 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, thereby making it possible to recover extracellular vesicles with a specific particle size range.

[0251] In the desorption step, when the recovery operation is repeated two or more times, the desorption time may be changed for each recovery operation. The desorption time for each recovery operation can be arbitrarily selected 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.

[0252] The method for purifying extracellular vesicles according to the second embodiment 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 salting out, precipitating and removing proteins using polymer flocculants or organic solvents, and adding a protein adsorbent.

[0253] The method for purifying extracellular vesicles according to the second embodiment may be capable of adsorbing 30% or more, 50% or more, 70% or more, or 80% or more of the extracellular vesicles contained in a serum-free medium sample. The method for purifying extracellular vesicles according to the second embodiment may be capable of adsorbing 20% ​​or more, 40% or more, 60% or more, or 70% or more of the extracellular vesicles contained in a serum-containing sample.

[0254] The method for purifying extracellular vesicles according to the second embodiment may be capable of desorbing 10% or more, 30% or more, 50% or more, or 70% or more of the extracellular vesicles captured by the solid phase carrier for a serum-free medium sample. The method for purifying extracellular vesicles according to the second embodiment may be capable of desorbing 10% or more, 20% or more, 40% or more, or 60% or more of the extracellular vesicles captured by the solid phase carrier for a serum-containing sample.

[0255] 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.

[0256] <Test Example 1> In Test Example 1, "room temperature" means 25±5°C.

[0257] [Elemental Analysis] Phosphorus, calcium, and oxygen were quantified by energy dispersive X-ray (EDS) analysis using JSM-F100+JED-2300 (manufactured by JEOL Ltd.) The EDS analysis was performed under the conditions of an acceleration voltage of 15 kV and detection elements B to U.

[0258] Carbon and hydrogen were quantified using an elemental analyzer (Fully Automatic Organic Trace Elemental Analyzer 2400IICHNS / O, manufactured by PerkinElmer).

[0259] [Zeta Potential] The zeta potential was measured at 25°C using a Zetasizer Nano ZS (manufactured by Malvern). The sample was measured as is without pH adjustment. The viscosity and relative dielectric constant of the dispersion medium were analyzed using the values ​​for pure water.

[0260] [Particle size distribution] The particle size distribution was measured using a laser diffraction / scattering particle size distribution analyzer LA-950V2 (manufactured by Horiba, Ltd.) with ion-exchanged water as the dispersion medium. The number-average particle size was taken as the average diameter. Furthermore, particle sizes were calculated from the smallest number to determine the cumulative frequency percentages up to 10%, 50%, and 90%. The 50% value was taken as the median diameter.

[0261] [XRD] Crystal structure analysis was performed by XRD using SmartLab (manufactured by Rigaku Corporation). The dried sample was crushed in a mortar and then measured. The position of the peak with the strongest diffraction intensity was taken as the peak position, and the peak width measured at the position where the intensity of this peak was half was taken as the half-width. XRD measurement conditions: SmartLab (rotating anode) manufactured by Rigaku Corporation; X-ray source: CuKα ray; Output: 45 kV, 200 mA; Optical system: focusing method (BB mode); Scan range: 5 to 80 deg.; Step width: 0.01 deg.; Scan speed: 10.00 deg. / min; Sample rotation: 10 mm; Detector: D / tex Ultra250 (1D mode).

[0262] [Wet Bulk Gravity (Dry Mass of Sediment Layer / Swelled Volume)] A dried sample of the precipitate (white solid) obtained in each of the Examples and Comparative Examples described below was dispersed in ion-exchanged water, allowed to stand for 1 hour, and then centrifuged at 5,000 g for 5 minutes to separate the precipitate into a clear supernatant (upper layer) and a cloudy sediment layer (lower layer), after which the supernatant was completely removed. The dry mass of the sediment layer / swelled volume (g / L) was calculated by dividing the mass of the white solid in the dry state (g) by the volume of the white solid in the swollen state (L), using the volume of the sediment layer (swollen white solid) and the mass of the white solid after drying.

[0263] [Total Light Transmittance and Haze] A dried sample of the precipitate was dispersed in ion-exchanged water, allowed to stand for 1 hour, and then centrifuged at 5000 g for 5 minutes to separate it into a clear supernatant (upper layer) and a cloudy sediment layer (lower layer), and the supernatant was completely removed. Subsequently, ion-exchanged water was added to the sediment layer in an amount 14 times its volume. Ultrasonic waves were irradiated for 1 minute to disperse the precipitate. This liquid was placed in a cell with an optical path length of 1 cm, and the total light transmittance and haze were measured using a spectroscopic haze meter SH7000 (manufactured by Nippon Denshoku Industries Co., Ltd.) with the cell containing ion-exchanged water as a reference.

[0264] [Example 1-1] Each component was dissolved in pure water and mixed to a final concentration of 2000 mg / L ammonium carbonate, 800 mg / L dipotassium hydrogen phosphate, and 200 mg / L calcium chloride. After mixing, the mixture was left to stand overnight at room temperature to precipitate a white solid. This was used as a slurry containing the solid phase carrier of Example 1-1.

[0265] The solubility of the white solid, the adsorption rate of extracellular vesicles, the washing loss rate, and the recovery rate were evaluated by the following methods.

[0266] [Solubility of White Solid] 1 mg of white solid was added to 1 mL of pure water or 500 mM EDTA aqueous solution, and the mixture was vigorously shaken for 30 seconds every 5 minutes at 20°C ± 5°C to confirm whether the solid was completely dissolved within 30 minutes.

[0267] The solid phase carrier (white solid) of Example 1-1 was a component that was insoluble in pure water but soluble in the aqueous solution of a chelating agent.

[0268] [Evaluation of extracellular vesicle adsorption rate] 0.1 mL of a liquid containing solid carriers was added to 0.1 mL of a medium (culture supernatant; a liquid containing extracellular vesicles) obtained by culturing cells in a serum-free medium for 3 days, and the mixture was stirred at room temperature for 10 seconds using a vortex mixer. After centrifugation at 5000 g, the supernatant was sucked up with a pipette, and the supernatant, from which the solid carriers had been removed by centrifugation, was collected and used as a measurement sample.

[0269] Using microRNA or CD63 as the measurement target, the concentration of extracellular vesicles was measured by evaluation using Qubit (Thermo Fisher Scientific) or an exosome ELISA kit, and the amount (number) of extracellular vesicles contained in the sample was determined. This amount was used to calculate the adsorption rate using the following formula. Note that when the value was below the detection limit, the measured value was set to 0, and the amount of extracellular vesicles was also set to 0. (Amount before addition of solid phase carrier - Amount after addition of solid phase carrier) x 100 / Amount before addition of solid phase carrier [%]

[0270] [Evaluation of washing loss rate of extracellular vesicles] Extracellular vesicles were adsorbed onto the surface of a solid phase carrier using the same procedure as in the evaluation of the adsorption rate of extracellular vesicles. The solid phase carrier with adsorbed extracellular vesicles was centrifuged at 5000 g, and then 0.1 mL of PBS(-) buffer was added to the solid phase carrier and washed by stirring for 10 seconds at room temperature using a vortex mixer. After centrifugation at 5000 g, the supernatant was sucked up with a pipette, and the supernatant, from which the solid phase carrier had been removed, was collected and used as a measurement sample. As in the evaluation of the adsorption rate of extracellular vesicles, the concentration of extracellular vesicles was determined using the concentration of microRNA or CD63 as the target, and the washing loss rate was calculated using the following formula: (amount of extracellular vesicles contained in solution) x 100 / amount before addition of solid phase carrier [%]

[0271] [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 a solid support, the solid support was separated, and then washed with PBS(-) buffer. The washed solid support with adsorbed extracellular vesicles was centrifuged at 5000 g, and 0.1 mL of 20 mmol / L EDTA solution was added and stirred for 10 seconds at room temperature using a vortex mixer. If the solid support was completely dissolved, this solution was used as the measurement sample. If some of the solid support was not dissolved, the solution containing the solid support and EDTA was centrifuged at 5000 g, and the supernatant was aspirated with a pipette. The supernatant excluding the solid support was used as the measurement sample. As with the evaluation of the extracellular vesicle adsorption rate, the concentration of extracellular vesicles was determined using the microRNA or CD63 concentration as the target, and the desorption rate was calculated using the following formula: (amount of extracellular vesicles contained in the solution) × 100 / amount before addition of solid phase carrier [%]

[0272] [Extracellular vesicle saturation adsorption test] 0.01-0.1 mL of liquid containing solid phase carriers was added to 1 mL of culture medium (culture supernatant; liquid containing extracellular vesicles) in which cells were cultured in serum-free medium for 3 days, and the mixture was stirred for 10 seconds at room temperature using a vortex mixer. After centrifugation at 5000 g, the supernatant was aspirated with a pipette, and the supernatant, from which the solid phase carriers had been removed, was collected and used as a measurement sample. The microRNA concentration was measured in the same manner as in the evaluation of the extracellular vesicle adsorption rate. The amount of microRNA adsorbed to the solid phase carrier was calculated using the following formula: (amount before addition of solid phase carriers - amount after addition of solid phase carriers) [ng]

[0273] However, if the amount after addition of the solid phase carrier was 10% or less of the amount before addition of the solid phase carrier, or was below the detection limit of the microRNA, the measurement was repeated under conditions in which the amount of solid phase carrier added was reduced.

[0274] The liquid containing the solid phase carrier was dried at room temperature and weighed to determine the dry weight of the solid phase carrier contained in the liquid containing the solid phase carrier. Using these values, the amount of microRNA adsorbed per unit weight of the solid phase carrier was calculated using the following formula: Amount of microRNA adsorbed to the solid phase carrier / Dry weight of added solid phase carrier [ng / mg]

[0275] [Low chelate desorption test of extracellular vesicles] Using the same procedures as those used to evaluate the adsorption rate of extracellular vesicles and the washing loss rate, extracellular vesicles were adsorbed onto the surface of a solid support and then washed. A 2 mmol / L EDTA solution was added and stirred at room temperature for 10 seconds. The supernatant obtained by removing the solid support from the solution was used as the measurement sample. As with the evaluation of the adsorption rate of extracellular vesicles, the concentration of extracellular vesicles was determined using the microRNA concentration as the target, and the desorption rate was calculated using the following formula: (amount of extracellular vesicles contained in the solution) x 100 / amount before addition of the solid support [%]

[0276] [Example 1-2] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 2,000 mg / L of ammonium carbonate and 800 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-2 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution. The calcium standard solution used in Example 1-2 and the following examples and comparative examples was a calcium standard solution (Ca: 1,000 mg / L) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., which is a solution prepared by dissolving calcium carbonate in nitric acid.

[0277] [Example 1-3] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of ammonium carbonate, 800 mg / L of tripotassium phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-3 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0278] [Example 1-4] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of ammonium carbonate, 800 mg / L of disodium hydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-4 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0279] [Example 1-5] The final concentrations of carbonate and phosphate were changed to 2000 mg / L magnesium carbonate and 800 mg / L trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L calcium carbonate. The solution was filtered before mixing the three types of reagents. Other operations were the same as in Example 1-1, and the solid phase carrier was prepared and evaluated. The solid phase carrier (white solid) in Example 1-5 was a component that was insoluble in pure water but soluble in a chelating agent aqueous solution.

[0280] [Example 1-6] The final concentrations of carbonate and phosphate were changed to 2000 mg / L magnesium carbonate and 800 mg / L tripotassium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L calcium carbonate. The solution was filtered before mixing the three reagents. Other operations were the same as in Example 1-1, and the solid phase carrier was prepared and evaluated. The solid phase carrier (white solid) in Example 1-6 was a component that was insoluble in pure water but soluble in a chelating agent aqueous solution.

[0281] [Example 1-7] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of tripotassium phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-7 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0282] [Example 1-8] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of disodium hydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-8 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0283] [Example 1-9] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-9 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0284] [Amount of EDTA required to dissolve the white solid] 1 mL of 1 mM to 100 mM EDTA aqueous solution was added to 1 mg of the white solid, and the EDTA concentration at which the white solid completely dissolved was determined. The EDTA concentration required to dissolve the white solid was determined as the average of the EDTA concentration at which the white solid completely dissolved and the EDTA concentration at which some EDTA remained undissolved, and the amount of EDTA per 1 g of the white solid was calculated.

[0285] [Particle Shape] The average diameter of the primary particles was observed using a transmission electron microscope (JEM-2100F manufactured by JEOL Ltd.), and the particle shape of the secondary particles was observed using a scanning electron microscope (VE-9800 manufactured by Keyence Corporation). The secondary particles were cluster-like particles formed by association of primary particles with an average diameter of 80 nm. The ratio of the average diameter of the secondary particles to the average diameter of the primary particles was 40.37.

[0286] [Example 1-10] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 500 mg / L of ammonium carbonate and 800 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-10 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0287] [Example 1-11] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 4000 mg / L of ammonium carbonate and 800 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-11 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0288] [Example 1-12] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 2000 mg / L of ammonium carbonate and 400 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-12 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0289] [Example 1-13] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 2000 mg / L of ammonium carbonate and 3200 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 500 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-13 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0290] [Example 1-14] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 2000 mg / L of ammonium carbonate and 800 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 250 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-14 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0291] [Example 1-15] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations of carbonate and phosphate were changed to 2000 mg / L of ammonium carbonate and 800 mg / L of trisodium phosphate, and a calcium standard solution was used so that the final concentration of calcium salt was 4000 mg / L of calcium carbonate. The solid phase carrier (white solid) in Example 1-15 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0292] [Example 1-16] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 100 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-16 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0293] [Example 1-17] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to sodium bicarbonate 500 mg / L, sodium dihydrogen phosphate 800 mg / L, and calcium chloride 200 mg / L. The solid phase carrier (white solid) in Example 1-17 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0294] [Example 1-18] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 1000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-18 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0295] [Example 1-19] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 4000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-19 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0296] [Example 1-20] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 8000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-20 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0297] [Example 1-21] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 100 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-21 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0298] [Example 1-22] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 400 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-22 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0299] [Example 1-23] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 1600 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-23 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0300] [Example 1-24] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 3200 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-24 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0301] [Example 1-25] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 6400 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-25 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0302] [Example 1-26] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 50 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-26 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0303] [Example 1-27] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 100 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-27 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0304] [Example 1-28] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 200 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-28 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0305] [Example 1-29] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 400 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-29 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0306] [Example 1-30] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 800 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-30 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0307] [Example 1-31] ​​A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 2000 mg / L of sodium bicarbonate, 800 mg / L of sodium dihydrogen phosphate, and 1600 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-31 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0308] [Example 1-32] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to sodium bicarbonate 500 mg / L, sodium dihydrogen phosphate 160 mg / L, and calcium chloride 40 mg / L. The solid phase carrier (white solid) in Example 1-32 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0309] [Example 1-33] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 1000 mg / L of sodium bicarbonate, 400 mg / L of sodium dihydrogen phosphate, and 100 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-33 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0310] [Example 1-34] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 4000 mg / L of sodium bicarbonate, 1600 mg / L of sodium dihydrogen phosphate, and 400 mg / L of calcium chloride. The solid phase carrier (white solid) in Example 1-34 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0311] [Example 1-35] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 800 mg / L dipotassium hydrogen phosphate and 200 mg / L calcium chloride. The solid phase carrier (white solid) in Example 1-35 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0312] [Example 1-36] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentration of the phosphate was changed to 800 mg / L trisodium phosphate and that a calcium standard solution was used so that the final concentration of the calcium salt was 500 mg / L calcium carbonate. The solid phase carrier (white solid) in Example 1-36 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0313] [Example 1-37] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 800 mg / L disodium hydrogen phosphate and 200 mg / L calcium chloride. The solid phase carrier (white solid) in Example 1-37 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0314] [Example 1-38] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 8 g / L of dipotassium hydrogen phosphate and 2 g / L of calcium chloride. The solid phase carrier (white solid) in Example 1-38 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0315] [Example 1-39] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 16 g / L of trisodium phosphate and 4 g / L of calcium chloride. The solid phase carrier (white solid) in Example 1-39 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0316] [Example 1-40] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 40 g / L of disodium hydrogen phosphate and 10 g / L of calcium chloride. The solid phase carrier (white solid) in Example 1-40 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0317] [Example 1-41] A solid phase carrier was prepared and evaluated in the same manner as in Example 1-1, except that the final concentrations were changed to 80 g / L trisodium phosphate and 20 g / L calcium chloride. The solid phase carrier (white solid) in Example 1-41 was a component that was insoluble in pure water but soluble in an aqueous chelating agent solution.

[0318] [Example 2-1] A white solid was produced in the same manner as in Example 1-1. The polymer magnetic particles synthesized by the following method were mixed with the white solid, and the white solid was immobilized on the surface of the polymer magnetic particles. This resulted in the solid phase carrier of Example 2, which had a white solid on the surface and contained a polymer (extracellular vesicle non-adhesive layer) as an intermediate layer. Except for using the solid phase carrier of Example 2, evaluation was performed in the same manner as in Example 1-1.

[0319] (Synthesis of Magnetic Particles) Step (i) (Micromagnetic 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) having 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.

[0320] Step (ii) (Drying and High-Speed ​​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 rotation speed of 11,300 min. －１ The treatment was carried out for 5 minutes at an average temperature of 35°C during the treatment.

[0321] Step (iii) (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, followed by a reaction at 60°C and 180 rpm for 3 hours. 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 styrene, 0.05 g of methacrylic acid, 0.15 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, followed by drying under reduced pressure.

[0322] [Example 2-2] A solid phase carrier having a white solid on the surface and a polymer (extracellular vesicle non-adhesive layer) as an intermediate layer was obtained in the same manner as in Example 2-1, except that 0.5 g of 2-methacryloyloxyethyl phosphorylcholine was used as the monomer per 1 g of particles.

[0323] Comparative Example 1 Tricalcium phosphate was used as a solid phase carrier, and a solution prepared by dispersing tricalcium phosphate in pure water at 3000 mg / L was used as a slurry.

[0324] Comparative Example 2 Calcium diphosphate was used as a solid phase carrier, and a solution prepared by dispersing calcium diphosphate in pure water at 3000 mg / L was used as a slurry.

[0325] Comparative Example 3 Hydroxyapatite was used as a solid support, and a solution prepared by dispersing hydroxyapatite in pure water at 3000 mg / L was used as a slurry.

[0326] Comparative Example 4 Dipotassium hydrogen phosphate was used as a solid phase carrier, and a solution prepared by dispersing it in pure water at 3000 mg / L was used as a slurry.

[0327] Comparative Example 5 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-35, except that dipotassium hydrogen phosphate was not added.

[0328] Comparative Example 6 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-35, except that calcium chloride was not added.

[0329] Comparative Example 7 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-36, except that trisodium phosphate was not added.

[0330] Comparative Example 8 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-36, except that calcium was not added.

[0331] Comparative Example 9 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-37, except that disodium hydrogen phosphate was not added.

[0332] Comparative Example 10 A solid phase carrier was prepared and evaluated in the same manner as in Example 1-37, except that calcium chloride was not added.

[0333] [Evaluation Results] Tables 1 to 7 show the evaluation results of the solubility of the white solid, the adsorption rate of extracellular vesicles, the washing loss rate, and the recovery rate. In the "Solubility" results in the tables, "-" indicates that no solid was generated and the solubility test could not be performed.

[0334]

[0335]

[0336] [Elemental Analysis Results] Elemental analysis was performed on the solid phase supports of the Examples and Comparative Examples shown in Table 8. Table 8 shows the measurement results of the molar ratios of various elements (carbon, oxygen, hydrogen, and calcium) to 1 mol of phosphorus.

[0337] [Zeta Potential Measurement Results] The zeta potential was measured for the solid phase supports of the Examples and Comparative Examples shown in Table 9. The results are shown in Table 9.

[0338] [Measurement Results of Bulk Specific Gravity] The bulk specific gravity (dry mass of sedimentation layer / swollen volume) in a wet state was measured for the solid phase carriers of the Examples shown in Table 10. The results are shown in Table 10.

[0339] It was confirmed that the solid phase carriers (white solids) of Examples 1-1 and 1-9 had high bulk specific gravity. From a comparison between Example 1-9 and Example 1-35, it can be seen that, for example, when the white solid contains carbon element, the bulk specific gravity becomes even higher.

[0340] [Measurement Results of Total Light Transmittance and Haze] The total light transmittance and haze were measured for the solid phase carriers of the Examples shown in Table 11. The results are shown in Table 11.

[0341] It was confirmed that the total light transmittance and haze can be adjusted by controlling the contained elements, particle size, etc. in the solid phase carrier (white solid).

[0342] [Measurement Results of Particle Size Distribution] The particle size distribution (average diameter, cumulative 10% diameter, cumulative 50% diameter, and cumulative 90% diameter) was measured for the solid phase carriers of the Examples shown in Table 12. The results are shown in Table 12.

[0343] [XRD Measurement Results] XRD measurements were performed on the solid phase supports of the Examples shown in Table 13. Table 13 shows the positions (2θ) and half-widths of the peaks with the highest intensity. Table 13 also shows the positions and intensities (PI) of the peaks located in the range of 30° to 35° in 2θ. １ ), the position and intensity of the peaks located in the range of 28° to less than 30° in 2θ (PI ２ ), the position and intensity of the peaks located in the range of 10° to 15° in 2θ (PI ３ ), and the intensity ratio of each peak (PI ２ / PI １ and P.I. ３ / PI １ ) is also shown.

[0344] [Results of the test to confirm the amount of EDTA required to dissolve the white solid] A test to confirm the amount of EDTA required to dissolve the white solid was performed using Examples 1-9 and 1-37. The evaluation results are shown in Table 14.

[0345] [Results of saturation adsorption test of extracellular vesicles] Table 15 shows the results of the saturation adsorption test of extracellular vesicles using Examples 1-1, 1-9, 1-37 and Comparative Example 3.

[0346] A comparison of the Examples and Comparative Examples shown in Table 15 confirmed that the use of the solid phase carriers of the Examples resulted in a higher adsorption amount in the saturated adsorption test. A comparison of Examples 1-9 and 1-37 shows that, for example, the use of a white solid (solid phase carrier) containing carbon element results in a higher adsorption amount in the saturated adsorption test.

[0347] [Results of low chelate desorption test for extracellular vesicles] Table 16 shows the results of low chelate desorption test for extracellular vesicles using the examples and comparative examples shown in the table below.

[0348] It was confirmed that the adsorption amount in the low chelate desorption test was further increased when the solid phase carriers of the Examples shown in Table 16 were used. These results show that, for example, when a white solid (solid phase carrier) containing carbon element is used, extracellular vesicles can be purified with a high recovery rate even when an aqueous chelating agent solution with a low chelating agent concentration is added.

[0349] <Reference Test Example: Test to confirm the effect of a chelating agent> [Reference Example 1] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, a 20 mmmol / L pyridine solution was used instead of the EDTA solution, and the evaluation was otherwise performed in the same manner as in Example 1-1.

[0350] [Reference Example 2] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, a hydrochloric acid solution of pH 6 was used instead of the EDTA solution, and the evaluation was otherwise performed in the same manner as in Example 1-1.

[0351] [Reference Example 3] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, ion-exchanged water was used instead of the EDTA solution, and the other procedures were the same as in Example 1-1.

[0352] [Reference Example 4] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, phosphate buffered saline (PBS buffer) at pH 7.4 was used instead of the EDTA solution, and the evaluation was otherwise performed in the same manner as in Example 1-1.

[0353] [Reference Example 5] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, a phosphate buffer solution of pH 8 was used instead of the EDTA solution, and the other procedures were the same as in Example 1-1.

[0354] [Reference Example 6] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, a Tris-HCl buffer solution of pH 9 was used instead of the EDTA solution, and the other procedures were the same as in Example 1-1.

[0355] [Reference Example 7] A solid phase carrier was prepared in the same manner as in Example 1-10. In evaluating the recovery rate of extracellular vesicles, an aqueous sodium hydroxide solution at pH 10 was used instead of the EDTA solution, and the other procedures were the same as in Example 1-1.

[0356] [Evaluation Results] Tables 17 and 18 show the results of a test to confirm the effect of the chelating agent. Tables 17 to 18 also show the test results for the solubility of the white precipitate in various test solutions. The test solutions used were EDTA (Examples 1 to 9), pyridine solution (Reference Example 1), aqueous hydrochloric acid solution (Reference Example 2), pure water (Reference Example 3), PBS buffer solution (Reference Example 4), phosphate buffer solution (Reference Example 5), Tris-hydrochloric acid buffer solution (Reference Example 6), and aqueous sodium hydroxide solution (Reference Example 7). The white precipitate dissolution test evaluated the solubility of the white precipitate in the same manner as described above in [Solubility of white solids], except that these test solutions were used.

[0357] It was confirmed that the recovery rate was further improved when a chelating agent was used in the purification of extracellular vesicles.

[0358] <Test Example 2> In Test Example 2, "room temperature" means 20±10°C.

[0359] [Evaluation of extracellular vesicle adsorption rate] Cells were cultured for 3 days in serum-free medium or medium supplemented with 10% exosome-free serum (culture supernatant; liquid containing extracellular vesicles), and 0.5 mL of an aqueous solution of an adsorption promoter was mixed with 0.5 mL. 10 mg of solid carrier was added to this mixture and stirred at room temperature for 1 hour. The supernatant, after removing the solid carrier from the solution, was used as the measurement sample.

[0360] Using microRNA as the measurement target, the concentration of extracellular vesicles was measured by evaluation using Qubit (Thermo Fisher Scientific), and the adsorption rate was calculated using the following formula: (concentration before addition of solid phase carrier - concentration after addition of solid phase carrier) x 100 / concentration before addition of solid phase carrier [%]

[0361] [Evaluation of washing loss rate of extracellular vesicles] Extracellular vesicles were adsorbed onto the surface of a solid phase carrier using the same procedure as in the evaluation of the adsorption rate of extracellular vesicles. After separating the solid phase carrier with adsorbed extracellular vesicles, 10 mmol / L Tris-HCl buffer (pH 9) was added and stirred at room temperature for 5 minutes. The supernatant obtained by removing the solid phase carrier from the solution was used as the measurement sample. As in the evaluation of the adsorption rate of extracellular vesicles, the concentration of extracellular vesicles was determined using the microRNA concentration as the target, and the washing loss rate was calculated using the following formula: (amount of extracellular vesicles contained in the solution) x 100 / concentration before addition of the solid phase carrier [%]

[0362] [Evaluation of extracellular vesicle recovery rate] Using the same procedures as those for evaluating the extracellular vesicle adsorption rate and washing loss rate, extracellular vesicles were adsorbed onto the surface of a solid phase carrier and then washed. After separating the solid phase carrier with adsorbed extracellular vesicles, a 1 mmol / L EDTA solution was added and stirred at room temperature for 1 hour. The supernatant obtained by removing the solid phase carrier from the solution was used as the measurement sample. As with the evaluation of the extracellular vesicle adsorption rate, the concentration of extracellular vesicles was determined using the microRNA concentration as the target, and the desorption rate was calculated using the following formula: (amount of extracellular vesicles contained in the solution) x 100 / concentration before addition of the solid phase carrier [%]

[0363] [Test 1 to Confirm the Effect of Using a Solid-Phase Carrier Having a Metal Oxide on the Surface] [Example 3-1] (Synthesis of Magnetic Particles) Step (i) (Micromagnetic 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 and 100 rpm. 1.2 g of a magnetic material (material: magnetite, particle diameter: 10 nm, product name: EMG607, manufactured by Ferrotec Corporation) having a cationic dispersant on its surface was added to this particle dispersion, and the mixture was allowed to react at room temperature and 100 rpm for 2 hours. The solids (particles) were filtered off and washed with pure water.

[0364] Step (ii) (Drying and High-Speed ​​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 rotation speed of 11,300 min. －１ The treatment was carried out for 5 minutes at an average temperature of 35°C during the treatment.

[0365] Step (iii) (Coating of magnetized particles with metal oxide) 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, and 0.4 g of tetraethyl orthosilicate was added, followed by stirring 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 found to be approximately 0.02 to 0.05 μm.

[0366] (Adsorption promoter) The following procedures were performed to achieve 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 then diluted with pure water to adjust the concentration. This aqueous solution was used as an adsorption promoter and mixed with a liquid containing extracellular vesicles at a volume ratio of 1:1.

[0367] (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.

[0368] Example 3-2 Evaluation was carried out in the same manner as in Example 3-1, except that sodium dihydrogen phosphate was used instead of disodium hydrogen phosphate as the adsorption promoter in Example 3-1.

[0369] (Evaluation results) The adsorption rate of extracellular vesicles was 83%, which was a high adsorption rate. The washing loss rate was 4%, which was low. The recovery rate was 42%, which was a high recovery rate.

[0370] Example 3-3 Evaluation was carried out in the same manner as in Example 3-1, except that sodium bicarbonate was not used as the adsorption promoter in Example 3-1.

[0371] (Evaluation Results) The adsorption rate of extracellular vesicles was 64%, and the recovery rate was 29%.

[0372] Example 3-4 Evaluation was carried out in the same manner as in Example 3-1, except that disodium hydrogen phosphate was not used as the adsorption promoter in Example 3-1.

[0373] (Evaluation Results) The adsorption rate of extracellular vesicles was 68%, and the recovery rate was 26%.

[0374] Example 3-5 Evaluation was carried out in the same manner as in Example 3-1, except that calcium chloride was not used as the adsorption promoter in Example 3-1.

[0375] (Evaluation Results) The adsorption rate of extracellular vesicles was 59%, and the recovery rate was 39%.

[0376] Example 3-6 Evaluation was carried out in the same manner as in Example 3-1, except that sodium chloride was used instead of calcium chloride in the adsorption promoter of Example 3-1.

[0377] (Evaluation Results) The adsorption rate of extracellular vesicles was 38%, and the recovery rate was 13%.

[0378] Comparative Example 3-1 The adsorption promoter of Example 3-1 was not used, and pure water was used instead.Other than that, evaluation was carried out in the same manner as in Example 3-1.

[0379] (Evaluation Results) The adsorption rate of extracellular vesicles was 53%, the washing loss rate was 38%, and the recovery rate was 11%.

[0380] Comparative Example 3-2 Evaluation was carried out in the same manner as in Example 3-1, except that disodium hydrogen phosphate and calcium chloride were not used as the adsorption promoters in Example 3-1.

[0381] (Evaluation Results) The adsorption rate of extracellular vesicles was 46%. The recovery rate was 9%, which was low.

[0382] Comparative Example 3-3 Evaluation was carried out in the same manner as in Example 3-1, except that sodium bicarbonate and calcium chloride were not used in the adsorption promoter of Example 3-1.

[0383] (Evaluation Results) The adsorption rate of extracellular vesicles was 62%. The recovery rate was 0%, which was low.

[0384] Comparative Example 3-4 Evaluation was carried out in the same manner as in Example 3-1, except that sodium hydrogen carbonate and disodium hydrogen phosphate were not used as the adsorption promoters in Example 3-1.

[0385] (Evaluation Results) The adsorption rate of extracellular vesicles was 63%. The recovery rate was 0%, which was low.

[0386] Examples 4-1 to 4-26 Evaluations were carried out in the same manner as in Example 3-1, except that the amounts of carbonate, phosphate, and calcium salt used were changed as shown in the table.

[0387] [Test 2 to confirm the effect when using a solid phase carrier having a metal oxide on the surface] [Example 5-1] Magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 3-1, and the following step (iii) was further carried out.

[0388] Step (iii) (Coating of magnetized particles with metal oxide) 1 g of the magnetized particles was dispersed in 9 mL of 2-propanol, and 0.8 g of tetraethyl orthotitanate was added, followed by stirring 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 mixture was washed with methanol and dried under reduced pressure. By the above method, magnetic particles with a micromagnetic material content of 0.59 g / g per 1 g of magnetic particles were obtained.

[0389] (Evaluation results) The adsorption rate of extracellular vesicles was 89%, which was a high adsorption rate. The washing loss rate was 8%, which was low. The recovery rate was 42%, which was a high recovery rate.

[0390] Example 5-2 Magnetized particles were synthesized in the same manner as in steps (i) and (ii) in Example 3-1, and the following step (iii) was further carried out.

[0391] Step (iii) (Coating of magnetized particles with metal oxide) The magnetized particles were sputtered for 8 hours using a mixture of 95% by mass of indium oxide and 5% by mass of tin oxide as a target, to form a coating layer of indium oxide and indium tin oxide on the surface of the particles. 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.

[0392] (Evaluation results) The adsorption rate of extracellular vesicles was 88%, which was a high adsorption rate. The washing loss rate was 2%, which was low. The recovery rate was 65%, which was a high recovery rate.

[0393] [Test to confirm the effect when the type of solid phase carrier is changed] [Example 6-1] A column packed with commercially available silica gel particles was used, and evaluation was carried out in the same manner as in Example 3-1.

[0394] (Evaluation results) The adsorption rate of extracellular vesicles was 98%, which was a high rate. The washing loss rate was 13%, which was low. The recovery rate was 85%, which was a high rate.

[0395] Example 6-2 (Preparation of Well Plate) A polycarbonate film coated with silicon dioxide by sputtering was attached to the bottom of a bottomless 24-well plate.

[0396] (Evaluation Method) 200 μL of a liquid containing extracellular vesicles was used per well of a 24-well plate. Agitation was performed by shaking, and the evaluation was carried out in the same manner as in Example 3-1.

[0397] (Evaluation results) The adsorption rate of extracellular vesicles was 78%, which was a high rate. The washing loss rate was 2%, which was low. The recovery rate was 41%, which was a high rate.

[0398] [Test to confirm the effect when using a solid phase carrier having a polymer on the surface] [Example 7-1] (Method) Magnetic particles were synthesized in the same manner as in Example 2-3, except that the metal oxide coating in Example 3-1 was not performed and the surface was coated with a polymer according to the following procedure. These were used as the magnetic particles of Example 7-1.

[0399] (Polymer Coating of 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, followed by a reaction at 60°C and 180 rpm for 3 hours. The particles were 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 particles were reacted at 80°C for 2 hours under a nitrogen atmosphere. The particles were washed with pure water and methanol, followed by drying under reduced pressure.

[0400] (Evaluation results) The adsorption rate of extracellular vesicles was 82%, which was a high adsorption rate. The washing loss rate was 12%, which was low. The recovery rate was 68%, which was a high recovery rate.

[0401] Magnetic particles of Comparative Example 7-1 were prepared using pure water instead of the adsorption promoter used in Example 7-1. Otherwise, evaluation was carried out using the same procedures as in Example 7-1.

[0402] (Evaluation Results) From a comparison between Example 7-1 and Comparative Example 7-1, it was confirmed that the adsorption rate, washing loss rate, and recovery rate were improved by using an adsorption promoter, even when the solid phase carrier had a polymer on its surface.

[0403] Regarding the results of "Extracellular vesicles (measured by CD63)" in the table, "-" indicates that the results have not been evaluated.

[0404]

[0405] 1...core part, 2...micromagnetic body, 3...metal oxide, 4, 5...surface layer, 6...intermediate layer, 10...solid phase carrier.

Claims

1. Extracellular vesicle purification kit containing components that are insoluble in pure water but soluble in aqueous chelating agent solutions.

2. The extracellular vesicle purification kit according to claim 1, wherein the components include carbon, phosphorus, and oxygen elements.

3. The extracellular vesicle purification kit according to claim 2, wherein the number of moles of carbon element in said component is 5.0 moles or less per mole of phosphorus element in said component.

4. The extracellular vesicle purification kit according to claim 2 or 3, wherein the number of moles of oxygen element in the component is 1.00 to 20.00 moles per mole of phosphorus element in the component.

5. The extracellular vesicle purification kit according to claim 2 or 3, wherein the components further comprise calcium element.

6. The extracellular vesicle purification kit according to claim 2 or 3, wherein the number of moles of calcium element in the component is 1.00 to 10.00 moles per mole of phosphorus element in the component.

7. The extracellular vesicle purification kit according to claim 2 or 3, wherein the component further contains a hydrogen element, and the number of moles of the hydrogen element in the component is 0.1 to 10.0 moles per mole of the phosphorus element in the component.

8. An extracellular vesicle purification kit described in any one of claims 1 to 3, wherein the component has the most intense peak in an X-ray diffraction spectrum within a 2θ value range of 30° or more and 35° or less.

9. An extracellular vesicle purification kit described in any one of claims 1 to 3, wherein the half-width of the most intense peak in the X-ray diffraction spectrum of the component is in the range of 0.2° or more and 5.5° or less.

10. An extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the components contain phosphate ions and calcium ions as constituent ions.

11. The extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the component is a reaction product of a carbonate, a phosphate, and a calcium salt.

12. An extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the ratio of the mass of the component in a dry state to the volume of the component in a swollen state is 10 to 100 g / L.

13. An extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the haze of the liquid in which the components are dispersed is 70 to 100%.

14. The extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the component is a cluster of secondary particles formed by the aggregation of primary particles, and the ratio of the average diameter of the secondary particles to the average diameter of the primary particles is 10 to 1000.

15. An extracellular vesicle purification kit according to any one of claims 1 to 3, wherein the component has peaks in an X-ray diffraction spectrum between 2θ values ​​of 30° or more and 35° or less and between 2θ values ​​of 28° or more and less than 30°, and the ratio of the intensity of the peaks in 2θ values ​​of 28° or more and less than 30° to the intensity of the peaks in 2θ values ​​of 30° or more and 35° or less is 0 to 0.

13.

16. The extracellular vesicle purification kit according to any one of claims 1 to 3, further comprising a chelating agent.

17. An extracellular vesicle purification kit according to any one of claims 1 to 3, comprising a solid support having the component on at least its surface.

18. The extracellular vesicle purification kit according to claim 17, wherein the solid phase carrier is a magnetic particle.

19. The extracellular vesicle purification kit according to any one of claims 1 to 3, comprising a solid support comprising: a core portion; an intermediate layer comprising an extracellular vesicle non-adhesive layer and covering at least a portion of the surface of the core portion; and a surface layer comprising the component and covering at least a portion of the surface of the intermediate layer.

20. A method for purifying extracellular vesicles using the extracellular vesicle purification kit according to any one of claims 1 to 3, comprising: an adsorption step of contacting a liquid containing the extracellular vesicles with the component to adsorb the extracellular vesicles to the component; a separation step of separating the component adsorbed by the extracellular vesicles from the liquid; a washing step of washing the component adsorbed by the extracellular vesicles; and a desorption step of detaching the extracellular vesicles from the component adsorbed by the extracellular vesicles.

21. A method for producing a material for extracellular vesicle purification, comprising the step of reacting a carbonate, a phosphate, and a calcium salt in an aqueous solution to obtain the material for extracellular vesicle purification as a white precipitate.

22. An extracellular vesicle purification kit comprising the following (A) and (B): (A) a solid phase carrier, and (B) a component comprising two or more ions selected from the group consisting of carbonate ions, phosphate ions, and calcium ions.

23. The extracellular vesicle purification kit of claim 22, further comprising a chelating agent.

24. The extracellular vesicle purification kit according to claim 22 or 23, wherein component (B) is a component containing carbonate ions, phosphate ions, and calcium ions.

25. The extracellular vesicle purification kit according to claim 22 or 23, wherein component (B) is a component containing a carbonate salt, a phosphate salt, and a calcium salt.

26. The extracellular vesicle purification kit according to claim 25, wherein the carbonate is sodium bicarbonate, the phosphate is sodium dihydrogen phosphate or disodium hydrogen phosphate, and the calcium salt is calcium chloride.

27. The extracellular vesicle purification kit according to claim 22 or 23, wherein the solid support has a metal oxide on its surface.

28. The extracellular vesicle purification kit of claim 27, 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.

29. The extracellular vesicle purification kit according to claim 22 or 23, wherein the solid phase carrier is a magnetic particle having a metal oxide on its surface.

30. The extracellular vesicle purification kit according to claim 22 or 23, wherein the solid phase carrier has a non-adhesive layer for extracellular vesicles on its surface.

31. A method for purifying extracellular vesicles using the extracellular vesicle purification kit described in claim 22 or 23, comprising: an adsorption step of mixing a liquid containing the extracellular vesicles with component (A) and component (B) and adsorbing the extracellular vesicles to the surface of component (A); a separation step of separating component (A) to which the extracellular vesicles have adsorbed from the liquid; a washing step of washing component (A) to which the extracellular vesicles have adsorbed; and a desorption step of detaching the extracellular vesicles from component (A) to which the extracellular vesicles have adsorbed.

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