Method for recovering contents from extracellular vesicles

Abstract

This invention provides a method for recovering EV contents at high concentration and high yield. [Solution] A method for recovering contents from extracellular vesicles, comprising steps (1) and (2), wherein step (1) is a step of contacting a biological sample with a crosslinked polymer to obtain a polymer gel on which extracellular vesicles are adsorbed; step (2) is a step of disrupting the extracellular vesicles adsorbed on the polymer gel and recovering the contents of the extracellular vesicles; and step (2) comprises a step of disrupting the extracellular vesicles adsorbed on the polymer gel using a disruption solution containing a surfactant (2A) and / or a step of disrupting the extracellular vesicles adsorbed on the polymer gel by ultrasonic vibration (2B).

Description

[Technical Field]

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

[0002] Extracellular vesicles (hereinafter also called "EVs") secreted by cells are vesicles measuring 30 to 1000 nm in size, surrounded by a lipid bilayer, and containing proteins, nucleic acids (RNA, etc.), etc. Their presence has been confirmed in various bodily fluids throughout the body. The proteins and nucleic acids contained within EVs reflect the state of the cell from which they are secreted, and are useful as biomarkers for evaluating various diseases. To evaluate diseases and other conditions using EV contents as biomarkers, a technology is needed to recover the contents at high concentration and with high yield. Examples of such technologies are known, such as those described in Patent Documents 1 and 2. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2022 / 211006 [Patent Document 2] International Publication No. 2020 / 090859 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Patent Document 1 describes a technique for separating extracellular molecules (EVs) such as exosomes after contacting a cross-linked polymer with a biological sample to obtain a polymer gel, and then adding a salt such as sodium chloride. While this technique allows for high recovery of intact EVs, further processing of the recovered EVs is necessary to evaluate the encapsulated substances present inside the vesicles, such as RNA and miRNA. Furthermore, the dilution of the eluate due to the shrinkage of water-absorbing cross-linked beads during the EV separation process makes it unsuitable for obtaining high concentrations of encapsulated substances. Patent Document 2 describes a technique for extracting miRNA, one of the EV encapsulants, by capturing EVs with a film-like device and then disrupting them with a disruption solution. However, the device used in this technique has an extremely small channel volume, making it unsuitable for recovering miRNA from samples of about 1 mL. Furthermore, since the device used in this technique is not intended for recovering encapsulants other than miRNA, there is a need for a method to comprehensively recover all EV encapsulants. The object of the present invention is to provide a method for recovering EV contents at high concentration and high yield. [Means for solving the problem]

[0005] The inventors of this invention arrived at this present invention as a result of diligent research to solve the above problems. In other words, the present invention is as follows: [1] A method for recovering contents from extracellular vesicles, comprising steps (1) and (2), wherein step (1) is a step of contacting a biological sample with a crosslinked polymer to obtain a polymer gel on which extracellular vesicles are adsorbed; step (2) is a step of disrupting the extracellular vesicles adsorbed on the polymer gel and recovering the contents of the extracellular vesicles, wherein step (2) comprises a step of disrupting the extracellular vesicles adsorbed on the polymer gel using a disruption solution containing a surfactant (2A) and / or a step of disrupting the extracellular vesicles adsorbed on the polymer gel by ultrasonic vibration (2B). [2] The method according to [1], wherein the crosslinked polymer is a crosslinked polymer comprising monomer units having an acidic group and / or a neutralizing base thereof, and contains a compound having at least one group selected from the group consisting of a cationic group and a hydroxyl group. [Effects of the Invention]

[0006] According to the present invention, it is possible to selectively separate extracellular vesicles from biological samples and recover the contents of the separated extracellular vesicles at high concentration and high yield. The recovered contents can be evaluated as biomarkers and used for the diagnosis of diseases, etc.

BEST MODE FOR CARRYING OUT THE INVENTION

[0007] The method for recovering the inclusion from the extracellular vesicles of the present invention is a method including step (1) and step (2). Step (1) is a step of bringing a biological sample into contact with a crosslinked polymer to obtain a polymer gel adsorbed with extracellular vesicles, and step (2) is a step of disrupting the extracellular vesicles adsorbed to the polymer gel and recovering the inclusion of the extracellular vesicles. In the present invention, step (2) includes step (2A) of disrupting the extracellular vesicles adsorbed to the polymer gel using a disrupting solution containing a surfactant and / or step (2B) of disrupting the extracellular vesicles adsorbed to the polymer gel by ultrasonic vibration.

[0008] <Step (1)> Step (1) is a step of bringing a biological sample into contact with a crosslinked polymer to obtain a polymer gel adsorbed with extracellular vesicles.

[0009] <Crosslinked polymer> ​​​​​​​​​​​​​ Examples of the monovalent metal include alkali metals such as lithium, sodium, and potassium. Examples of the divalent metal include alkaline earth metals such as magnesium, calcium, and barium, lead, zinc, and tin.

[0013] The formula: NR4 + In the cation represented by the formula, when R is a hydrocarbon group which may be substituted, the hydrocarbon group may be, for example, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. Examples of the alkyl group include C1-6 alkyl groups such as methyl group, ethyl group, propyl group (n-propyl group or isopropyl group), butyl group (n-butyl group, isobutyl group, sec-butyl group, or t-butyl group). Examples of the cycloalkyl group include C5-14 cycloalkyl groups such as cyclopentyl group and cyclohexyl group. Examples of the aryl group include C6-14 aryl groups such as phenyl group and naphthyl group. Examples of the aralkyl group include C7-14 aralkyl groups such as benzyl group and phenethyl group.

[0014] Examples of the substituent that can substitute the hydrocarbon group include halogen atom, hydroxyl group, mercapto group, etc.

[0015] The formula: NR4 + In the cation represented by the formula, when two or three Rs form a ring together with the adjacent nitrogen atom, the ring may be a monocyclic ring such as a pyridine ring or an imidazole ring, or a condensed ring such as a quinoline ring. The ring may have one or more substituents, and examples of the substituent include halogen atom, hydroxyl group, amino group, alkyl group, haloalkyl group, hydroxyalkyl group, N,N-dialkylamino group, etc. The number of the substituents is, for example, 1, 2, or 3.

[0016] Examples of monomers having an acidic group include unsaturated carboxylic acids. Unsaturated carboxylic acids include, for example, unsaturated monocarboxylic acids and unsaturated dicarboxylic acids. Examples of unsaturated monocarboxylic acids include unsaturated monocarboxylic acids having 3 to 10 carbon atoms, such as acrylic acid, methacrylic acid, and crotonic acid. Examples of unsaturated dicarboxylic acids (including anhydrides in this specification) include unsaturated dicarboxylic acids having 4 to 10 carbon atoms, such as maleic acid, fumaric acid, citraconic acid, and itaconic acid, and their anhydrides.

[0017] Examples of monomers having acidic groups other than those mentioned above include (meth)acrylic monomers having a sulfonic acid group [for example, (meth)acrylic acid sulfoalkyl esters such as 2-(meth)acrylic acid sulfoethyl, and N-sulfoalkyl (meth)acrylamides such as 2-(meth)acrylamido-2-methylpropanesulfonic acid], and (meth)acrylic monomers having a phosphate group [for example, (meth)acrylic acid phosphonooxyalkyl esters such as 2-((meth)acryloyloxy)ethyl phosphate]. In this specification, (meth)acrylic means acrylic and / or methacrylic, and (meth)acryloyloxy means acryloyloxy and / or methacryloyloxy.

[0018] The monomer having an acidic group may be a single type or a combination of two or more types.

[0019] Examples of monomers having a neutralizing base for an acidic group include unsaturated carboxylate salts. Examples of unsaturated carboxylate salts include alkali metal salts of unsaturated carboxylic acids (e.g., sodium salts, potassium salts), alkylamine salts (e.g., trialkylamine salts such as triethylamine salt), alkanolamine salts (e.g., dialkanolamine salts such as diethanolamine salt, trialkanolamine salts such as triethanolamine salt), ammonium salts, or tetraalkylammonium salts (e.g., tetramethylammonium salt, tetraethylammonium salt).

[0020] Examples of monomers having a neutralizing base for acidic groups other than those mentioned above include neutralized salts of (meth)acrylic monomers having sulfonic acid groups and neutralized salts of (meth)acrylic monomers having phosphate groups. Examples of these neutralized salts include the alkali metal salts mentioned above.

[0021] The monomer having a neutralizing base for the acidic group may be a single type or a combination of two or more types.

[0022] In the present invention, it is preferable that the crosslinked polymer contains both monomer units having acidic groups and monomer units having neutralizing bases for the acidic groups. That is, in a crosslinked polymer containing monomer units having acidic groups, it is preferable that a portion of the monomer units having acidic groups are neutralized and replaced with monomer units having neutralizing bases for the acidic groups. The degree of neutralization [100 × (number of moles of neutralizing base for acidic groups) / (total number of moles of acidic groups and their neutralizing bases)] is not particularly limited, but may be, for example, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, or 50 mol% or more, and may also be 90 mol% or less, 85 mol% or less, or 80 mol% or less.

[0023] The crosslinked polymer may contain other monomer units in addition to monomer units having an acidic group and / or a neutralizing base thereof. Examples of other monomers include the following monofunctional ethylenically unsaturated monomers. • Esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) (e.g., alkyl esters such as methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, heptyl ester, octyl ester, 2-ethylhexyl ester; hydroxyalkyl esters such as 2-hydroxyethyl ester; haloalkyl esters such as 2,2,2-trifluoroethyl ester; aminoalkyl esters such as 2-aminoethyl ester; (mono or dialkylamino)alkyl esters such as 2-(N,N-dimethylamino)ethyl ester; cycloalkyl esters such as cyclohexyl ester; aryl esters such as phenyl ester, naphthyl ester; aralkyl esters such as benzyl ester, phenethyl ester; glycidyl ester; polyethylene glycol ester, etc.) • Unsaturated carboxylic acid amides (e.g., free amides; N-substituted amides such as N-monoalkylamides and N,N-dialkylamides) • Unsaturated dicarboxylic acid imides (e.g., maleimide, citracomimide, itaconimide, and their N-alkyl substituted, N-cycloalkyl substituted, or N-aryl substituted derivatives) • Alkenyl esters (e.g., vinyl esters, allyl esters, etc.) of saturated carboxylic acids (e.g., acetic acid, propionic acid, etc.) • Esters or amides of unsaturated sulfonic acids (for example, esters or amides of unsaturated sulfonic acids corresponding to the esters or amides of unsaturated carboxylic acids exemplified above) • Unsaturated alcohols (e.g., allyl alcohol, propenyl alcohol, etc.) • Unsaturated ethers (e.g., alkyl vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; alkyl allyl ethers such as methyl allyl ether and ethyl allyl ether; cycloalkyl vinyl ethers such as cyclohexyl vinyl ether; glycidyl vinyl ether, etc.) • Unsaturated nitriles (e.g., acrylonitrile, methacrylonitrile, etc.) • Olefins (e.g., ethylene, propylene, butene, pentene, hexene, etc.) • Aromatic vinyl compounds (e.g., styrene, α-methylstyrene, vinyltoluene, hydroxystyrene, etc.) • Heterocyclic vinyl compounds (e.g., N-vinylpyrrolidone, etc.) The other monomers may be a single type or a combination of two or more types.

[0024] The monomers constituting the crosslinked polymer preferably have a solubility in water at 25°C (g / 100g of water) of 1g or more, and more preferably 5g or more. Furthermore, the monomers constituting the crosslinked polymer may also be miscible with water.

[0025] A crosslinked polymer is a polymer having a crosslinked structure [a structure in which the constituent atoms of multiple linear polymers (polymers of monomers having acidic groups and / or neutralizing bases thereof) are covalently bonded to each other, either directly or via other atoms]. The crosslinked structure may be self-crosslinked if the monomers constituting the crosslinked polymer have reactive groups (for example, a combination of monomers having carboxyl groups and monomers having amino groups), but may also be crosslinked with any crosslinking agent as needed.

[0026] Examples of crosslinking methods using crosslinking agents include the following: 1) Method using the following internal crosslinking agent: A crosslinked polymer having a crosslinked structure is obtained by polymerizing the above-mentioned monomer and the internal crosslinking agent [for example, the method described in Japanese Patent Publication No. 2003-225565 and Japanese Patent Publication No. 2005-75982]. 2) Method using the following surface crosslinking agent: A polymer having the above monomer as an essential constituent monomer is crosslinked using a surface crosslinking agent to obtain a crosslinked polymer [for example, the methods described in Japanese Patent Publication No. 3648553, Japanese Unexamined Patent Publication No. 2003-165883, Japanese Unexamined Patent Publication No. 2003-225565, Japanese Unexamined Patent Publication No. 2005-75982, Japanese Unexamined Patent Publication No. 2005-95759, etc.]

[0027] The aforementioned crosslinking agent (including internal crosslinking agents and surface crosslinking agents) is not particularly limited, but examples include the following bifunctional or more ethylenically unsaturated monomers. Alkenyl ethers of dihydric or higher alcohols (e.g., vinyl ethers, allyl ethers, etc.) (e.g., vinyl ethers, allyl ethers, etc.) • Esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) and dihydric or higher alcohols (e.g., those exemplified in the ethers above) • Alkenyl esters (e.g., vinyl esters, allyl esters, etc.) of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) • Alkenyl esters (e.g., vinyl esters, allyl esters, etc.) of polycarboxylic acids (e.g., tartaric acid, citric acid, adipic acid, etc.) • Alkenyl esters of isocyanuric acid (e.g., triallyl isocyanate) • Alkylene bisacrylamide (e.g., methylene bisacrylamide) • Alkylene bismethacrylamide (e.g., methylene bismethacrylamide) • Di- or trialkenylamines (e.g., diallylamine, triallylamine, etc.) • Aromatic polyvinyl compounds (e.g., divinylbenzene)

[0028] As the aforementioned crosslinking agent, a crosslinking agent having at least two functional groups that can react with substituents (carboxyl groups, hydroxyl groups, etc.) of the monomer may also be used. As such a crosslinking agent, polyhydric alcohol glycidyl ethers (for example, alkylene glycol diglycidyl ethers such as ethylene glycol diglycidyl ether, alkanetriol di or triglycidyl ethers such as glycerin diglycidyl ether) can be used.

[0029] The crosslinking agent may be a single type or a combination of two or more types.

[0030] The amount of crosslinking agent can be appropriately selected according to the desired crosslinking density. For example, it may be 0.005 mol% or more, or 0.01 mol% or more, or 0.5 mol% or less, or 0.4 mol% or less, relative to the monomers constituting the crosslinked polymer.

[0031] The crosslinked polymer preferably contains (or supports) a compound (hereinafter referred to as "compound X") having at least one group selected from the group consisting of cationic groups and hydroxyl groups. That is, the crosslinked polymer preferably exists in the form of a coexistence with compound X. The crosslinked polymer preferably has compound X on its surface, more preferably compound X is attached or fixed to the surface of the crosslinked polymer by non-covalent bonds such as hydrogen bonds and ionic bonds, and even more preferably part or all of the surface of the crosslinked polymer is covered with compound X. The surface of the crosslinked polymer means the molecular chain of the crosslinked polymer, or, if the crosslinked polymer is in particulate or pellet form, the surface of the particles, pellets, etc. of the crosslinked polymer.

[0032] The presence of compound X in a crosslinked polymer can be identified, for example, by immersing the crosslinked polymer in a suitable solvent (e.g., a hydrocarbon solvent such as hexane), releasing compound X into the solvent, isolating compound X, and subjecting it to NMR and gas chromatography.

[0033] Compound X preferably has at least one group selected from the group consisting of cationic groups and hydroxyl groups, and an optionally substituted hydrocarbon group having 2 to 30 carbon atoms; more preferably has at least one group selected from the group consisting of cationic groups and hydroxyl groups, and an optionally substituted hydrocarbon group having 12 to 30 carbon atoms; and even more preferably has an optionally substituted hydrocarbon group having 12 to 30 carbon atoms at one end and at least one group selected from the group consisting of cationic groups and hydroxyl groups at the other end. In compound X, the number of hydrocarbon groups, cationic groups, or hydroxyl groups may be one or two or more. In this specification, even if compound X has a cationic group and a hydroxyl group is included in the cationic group, compound X shall be deemed to have both a cationic group and a hydroxyl group. Similarly, even if compound X has a hydrocarbon group having 2 to 30 carbon atoms (or 12 to 30 carbon atoms) which may be substituted, and a hydroxyl group is included as a substituent in the hydrocarbon group, compound X shall be deemed to have both the hydrocarbon group and the hydroxyl group.

[0034] The hydrocarbon group having 12 to 30 carbon atoms may be any of the following: an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group; an alicyclic hydrocarbon group such as a cycloalkyl group or a cycloalkenyl group; or an aromatic hydrocarbon group. Examples of substituents on the hydrocarbon group include a hydroxyl group, an oxo group (=O), an alkoxy group, an alkenyloxy group, or combinations thereof. The number of substituents on the hydrocarbon group may be, for example, one, two, three, four, or five.

[0035] The aliphatic hydrocarbon group having 12 to 30 carbon atoms may be either saturated or unsaturated. Examples include aliphatic hydrocarbon groups having 12 to 20 carbon atoms such as lauryl, myristyl, palmityl, stearyl, oleyl, linoleyl, and arachidyl groups.

[0036] For example, lecithin has a C12-30 acyl group as an aliphatic hydrocarbon group having 12 to 30 carbon atoms which may be substituted, and specifically has an oleoyloxy group and a palmitoyloxy group. More specifically, lecithin has a diacylglycerol skeleton having the acyl group.

[0037] Examples of the alicyclic hydrocarbon group having 12 to 30 carbon atoms include a group having a steroid skeleton.

[0038] Examples of the aromatic hydrocarbon group having 12 to 30 carbon atoms include fluorene, anthracene, phenanthrene, tetracene, pyrene, triphenylene, chrysene, tetraphenylene and the like.

[0039] The hydrocarbon group having 12 to 30 carbon atoms which may be substituted is preferably a group having a di-C12-30 acylglycerol skeleton or a group having a steroid skeleton.

[0040] In this specification, the "cationic group" includes a group that itself forms a cation and a group that does not itself form a cation but can form a cation by bonding with a proton. Examples of the cationic group include -N(R 1 )2 (where each R 1 is independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an alicyclic hydrocarbon group having 2 to 6 carbon atoms or an aromatic hydrocarbon group that forms a ring together with the adjacent nitrogen atom with two R 1 ), -N + (R 2 )3 (where each R 2 is independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an alicyclic hydrocarbon group having 2 to 6 carbon atoms or an aromatic hydrocarbon group that forms a ring together with the adjacent nitrogen atom with two R 2 ), and the like.

[0041] R 1 and R 2Examples of hydrocarbon groups having 1 to 6 carbon atoms include alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, and hexyl groups. Examples of substituents that can be substituted for hydrocarbon groups having 1 to 6 carbon atoms include hydroxyl, oxo, carboxylic acid, and sulfonic acid groups.

[0042] R in the above formula 1 and R 2 Examples of alicyclic hydrocarbon groups having 2 to 5 carbon atoms include alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups, and alkenyl groups obtained by unsaturating these groups, but are not limited to these. Examples of substituents that can be substituted for alicyclic hydrocarbon groups having 2 to 5 carbon atoms include hydroxyl, oxo, amino, carboxylic acid, and sulfonic acid groups.

[0043] R in the above formula 1 and R 2 Examples of aromatic hydrocarbon groups include monocyclic rings such as pyridine rings and imidazole rings, and fused rings such as quinoline rings. The ring may have one or more substituents, such as hydroxyl groups, amino groups, alkyl groups, haloalkyl groups, and hydroxyalkyl groups.

[0044] The above formula: -N(R 1 The group represented by )2 is preferably -NH2 or -N(R 11 )2(wherein, R 11 (wherein represents a hydroxyalkyl group having 1 to 5 carbon atoms). Examples of the hydroxyalkyl group having 1 to 5 carbon atoms include a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, and a hydroxypentyl group (including a pentahydroxypentyl group, etc.).

[0045] The above formula: -N + (R 2 The group represented by )3 is preferably R 2 -N is an aliphatic hydrocarbon group. + (CH3)3, -N+ (CH3)2(C2H5) and -N + (CH3)2(C3H7), and two Rs 2 A pyridinium group, an N-methylimidazolium group, etc., which are aromatic hydrocarbon groups having 2 to 6 carbon atoms that form a ring together with an adjacent nitrogen atom. These groups may be substituted with, for example, -CO2-, -SO3-, etc.

[0046] Preferred compounds of compound X include compounds having an aliphatic hydrocarbon group and a cationic group with 12 to 30 carbon atoms (for example, lecithin, hexadecyltrimethylammonium chloride, hexadecylpyridinium chloride, 1-hexadecyl-3-methylimidazolium chloride), etc.

[0047] From the viewpoint of improving the adsorption amount of extracellular vesicles, the lower limit of the chemical formula weight or the number average molecular weight of compound X is preferably 200 or more. The upper limit of the chemical formula weight or the number average molecular weight of compound X is preferably 10,000 or less. The number average molecular weight of compound X can be measured using gel permeation chromatography (GPC) under the following conditions, etc. <GPC measurement conditions> [1] Apparatus: Gel permeation chromatography [HLC-8120GPC, manufactured by Tosoh Corporation] [2] Columns: "TSKgel G6000PWxl" and "TSKgel G3000PWxl" (both manufactured by Tosoh Corporation) are connected in series. [3] Eluent: A solution prepared by dissolving 0.5 wt% of sodium acetate in methanol / water = 30 / 70 (volume ratio). [4] Standard substance: Polyethylene glycol (hereinafter abbreviated as PEG) [5] Injection conditions: Sample concentration 0.25 wt%, column temperature 40 °C

[0048] In the present invention, the crosslinked polymer is preferably a particle with a volume-average particle diameter in the range of 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, or 150 μm or more, in order to reduce impurities (such as albumin) in the separation of extracellular vesicles from biological samples. Furthermore, in the present invention, the crosslinked polymer is preferably a particle with a volume-average particle diameter in the range of 2000 μm or less, 1500 μm or less, 1000 μm or less, 500 μm or less, or 300 μm or less, from the viewpoint of improving the adsorption amount of extracellular vesicles. The volume-average particle diameter can be measured by the method described in the examples below.

[0049] In the present invention, the pH of a physiological saline solution containing 0.5% by weight of a crosslinked polymer based on the weight of physiological saline (hereinafter abbreviated as "physiological saline solution pH") is not particularly limited. In the separation of extracellular vesicles from biological samples, the physiological saline solution pH is preferably 6.5 or higher, 7 or higher, or 7.2 or higher, and preferably 8.5 or lower, 8 or lower, 7.9 or lower, or 7.5 or lower, in order to reduce negatively charged impurities (such as albumin). The physiological saline solution pH can be measured by the method described in the examples below.

[0050] The pH of a physiological saline solution can be adjusted by the degree of neutralization. If the pH of the physiological saline solution is too low, increasing the degree of neutralization tends to raise the pH, and if the pH of the physiological saline solution is too high, decreasing the degree of neutralization tends to lower the pH.

[0051] In the present invention, the water absorption ratio of the crosslinked polymer in physiological saline (absorption ratio of physiological saline) is preferably 1 g / g or more, more preferably 3 g / g or more, particularly preferably 5 g / g or more, and most preferably 10 g / g or more, from the viewpoint of improving the amount of extracellular vesicles adsorbed. In the present invention, if the water absorption ratio of the crosslinked polymer in physiological saline (absorption ratio of physiological saline) is too low, the water absorption ratio can be increased by reducing the amount of the crosslinking agent. In the present invention, the upper limit of the water absorption ratio of the crosslinked polymer in physiological saline (absorption ratio of physiological saline) is not particularly limited, but for example, it is preferably 120 g / g or less, more preferably 100 g / g or less, and particularly preferably 50 g / g or less. The absorption ratio of physiological saline can be measured by the method described in the examples below.

[0052] In the present invention, when the crosslinked polymer has cationic groups, the molar concentration of the cationic groups is 6 × 10⁻⁶, based on the weight of the crosslinked polymer after drying, from the viewpoint of improving the adsorption amount of extracellular vesicles. -6 ~1 × 10 -4 It is preferable that the concentration is mol / g. The weight of the cross-linked polymer after drying can be measured, for example, by the following method. The weight of the residue after placing 1 g of crosslinked polymer in a petri dish, covering it with filter paper, and heating and drying it in a circulating air dryer at 130°C for 60 minutes can be used as the weight of the crosslinked polymer after drying.

[0053] In the present invention, compound X contained in the crosslinked polymer exhibits excellent affinity for extracellular vesicles; therefore, the crosslinked polymer can be suitably used in the application of separating extracellular vesicles from biological samples.

[0054] In the present invention, a crosslinked polymer can be produced by a method comprising the steps of polymerizing (solution polymerization, emulsion polymerization, suspension polymerization, etc.) a composition containing a monomer, a crosslinking agent, a solvent, an initiator, and optionally a neutralizing agent, drying the polymer, and optionally classifying the polymer.

[0055] The biological sample used in step (1) is not particularly limited as long as it contains extracellular vesicles. Examples of biological samples include bodily fluids such as human or animal blood, plasma, serum, tears, saliva, breast milk, pleural fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, and urine; liquefied tissues or cells such as organs, hair, nails, skin, muscles, and nerves (including cell culture media and their supernatants); and extracts from plants. The biological sample may be a single type or a combination of two or more types.

[0056] In step (1), the amount of crosslinked polymer used is preferably, for example, 1 part by mass or more, 5 parts by mass or more, or 10 parts by mass or more, and preferably 1000 parts by mass or less, 500 parts by mass or less, or 100 parts by mass or less, per 1000 parts by mass of biological sample.

[0057] The temperature at which the crosslinked polymer is brought into contact with the biological sample is, for example, within the range of 1 to 35°C, and preferably within the range of 5 to 30°C. Furthermore, it is preferable to allow the sample to stand for a predetermined time after contact (for example, 10 minutes or more, 30 minutes or more, or 5 hours or less, or 4 hours or less).

[0058] Step (1) allows extracellular vesicles to be adsorbed onto the polymer gel. The adsorption of extracellular vesicles onto the polymer gel includes not only the form in which they are attached to the surface of the polymer gel, but also the form in which some or all of the extracellular vesicles are embedded inside the polymer gel.

[0059] Step (1) may include a step of washing the polymer gel after the extracellular vesicles have been adsorbed (washing step). The washing step can be performed, for example, by adding saline solution to the polymer gel after the extracellular vesicles have been adsorbed.

[0060] <Process (2)> Step (2) is a step of disrupting extracellular vesicles adsorbed onto a polymer gel and recovering the contents of the extracellular vesicles. Step (2) includes a step (2A) of disrupting the extracellular vesicles adsorbed onto the polymer gel using a disruption solution containing a surfactant and / or a step (2B) of disrupting the extracellular vesicles adsorbed onto the polymer gel by ultrasonic vibration.

[0061] From the viewpoint of ease of operation, it is preferable that step (2) includes step (2A) of disrupting extracellular vesicles adsorbed on the polymer gel using a disruption solution containing a surfactant. If step (2) includes both step (2A) and step (2B), either step may be performed first.

[0062] Examples of surfactants included in the crushed liquid used in step (2A) are anionic surfactants such as sodium dodecyl sulfate and sodium deoxycholate, nonionic surfactants such as polyoxyethylene sorbitan monolaurate and polyoxyethylene octylphenyl ether, and amphoteric surfactants such as 3-[(3-collamidopropyl)dimethylammonio]propanesulfonate, but are not limited to these.

[0063] From the viewpoint of recovering the amount of encapsulated material, it is preferable that the surfactant included in the crushed liquid contains at least one selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene octylphenyl ether, and 3-[(3-collamidopropyl)dimethylammonio]propanesulfonate.

[0064] The surfactant contained in the crushed liquid may be a single type or a combination of two or more types. From the viewpoint of water solubility, the surfactant preferably has an HLB value of 10 or higher, more preferably 12 or higher, and even more preferably 14 or higher. The HLB value is an indicator that shows the balance between hydrophilicity and lipophilicity, and is a value calculated by the Oda method described on page 212 of "Introduction to Surfactants" [published by Sanyo Chemical Industries, Ltd. in 2007, written by Takehiko Fujimoto].

[0065] The concentration of the surfactant in the lysate is preferably 0.01% by weight or more, and more preferably 0.1% by weight or more, from the viewpoint of dissolving extracellular vesicles. Furthermore, from the viewpoint of preventing adverse effects on the analysis of the recovered encapsulated material, it is preferably 10% by weight or less, and more preferably 2% by weight or less.

[0066] The crushed liquid may contain salts. Examples of salts include, but are not limited to, monovalent metal salts such as sodium salts and potassium salts. Examples of counteranions to salts include, but are not limited to, halide ions such as chloride ions and bromide ions, carbonate ions, bicarbonate ions, sulfate ions, bisulfate ions, phosphate ions, hydrogen phosphate ions, and dihydrogen phosphate ions.

[0067] The salt in the crushed liquid may be a single type or a combination of two or more types. The salt concentration of the crushed liquid is preferably 0.15 mol / L or higher in molar concentration from the viewpoint of preventing water absorption by the cross-linked polymer, but is not limited to this. The pH of the crushed liquid is preferably 6 or higher, more preferably 7 or higher. It is also preferably 9 or lower, and more preferably 8 or lower.

[0068] Commercially available lysate may be used. Examples of commercially available products that can be used include RIPA Buffer [manufactured by Nacalai Tesque Co., Ltd.] and RNA purification lysis buffer [Buffer MLP, manufactured by MACHEREY-NAGEL Co., Ltd.].

[0069] The mixing temperature of the polymer gel on which extracellular vesicles have been adsorbed and the lysate is preferably within the range of 1 to 35°C or 5 to 30°C. Furthermore, it is preferable to let it stand for a predetermined time after mixing (for example, 5 minutes or more, 30 minutes or more, or 5 hours or less, or 3 hours or less).

[0070] By mixing the polymer gel to which extracellular vesicles are adsorbed with a lysate, the extracellular vesicles are disrupted, and the substances contained within them (encapsulated substances) are eluted outside the extracellular vesicles. The encapsulated substances of the extracellular vesicles can be recovered by separating the lysate containing the encapsulated substances using a method appropriate to the type of encapsulated substance. When recovering RNA as an encapsulated substance from a lysate containing encapsulated substances, for example, the lysate can be added to a NucleoSpin miRNA Column (manufactured by MACHEREY-NAGEL), and then the RNA can be recovered and purified by following the protocol for NucleoSpin miRNA Plasma (manufactured by MACHEREY-NAGEL).

[0071] By performing step (2), the contents of extracellular vesicles adsorbed onto the polymer gel can be recovered in high quantities. Examples of recovered extracellular vesicle contents include membrane proteins present in the lipid bilayer of the extracellular vesicle, proteins present inside the vesicle, nucleic acids such as DNA and RNA, glycans, lipids, and other small molecules contained in the extracellular vesicle.

[0072] According to the present invention, encapsulated materials can be recovered in a high yield compared to conventional methods (such as extracellular vesicle purification methods and encapsulated material recovery methods). [Examples]

[0073] The present invention will be further described by the following examples, but the present invention is not limited thereto.

[0074] <Method for measuring the pH of physiological saline solution> 0.5 g of the sample was placed in a 100 mL cylindrical beaker with a diameter of 50 mm, and physiological saline (salt concentration 0.9 wt%) was added to make a total volume of 100 g. The mixture was stirred at 25°C at 60 rpm with a stirrer tip (length 30 mm) for 30 minutes, and then allowed to stand at 25°C for 1 minute. After standing, the pH of the supernatant at 25°C was measured using a pH meter [Horiba, Ltd., F-74] and this value was taken as the pH of the physiological saline solution.

[0075] <Method for measuring the absorption rate of physiological saline> A tea bag (20 cm long, 10 cm wide) made from nylon mesh with a mesh size of 63 μm (JIS Z8801-1:2006) was placed in the tea bag and immersed in 1000 ml of physiological saline (0.9 wt%) for 1 hour without stirring. After immersion, the tea bag was removed from the physiological saline and hung to drain for 15 minutes, after which the weight of the tea bag (h1) was measured. Furthermore, the weight of the tea bag (h2) was measured by performing the same procedure as for measurement (h1), except that the measurement sample was not used. The absorption ratio of physiological saline was determined by applying the measurement results of (h1) and (h2) to the following formula (1). The temperature of the physiological saline and the measurement atmosphere used was 25℃ ± 2℃. Absorption rate of physiological saline (g / g) = (h1) - (h2) (1)

[0076] <Manufacturing Example 1: Manufacturing of Crosslinked Polymer (A-1)> In a 1-liter beaker, 116.5 g of acrylic acid, 272.2 g of deionized water, and 3.0 g of ethylene glycol diglycidyl ether (2.6 parts by weight per 100 parts by weight of acrylic acid) were added and mixed to dissolve the crosslinking agent. While cooling the beaker in an ice bath, 96.1 g of 48.5% by weight sodium hydroxide aqueous solution was added to neutralize a portion of the acrylic acid (72 mol%). After the neutralized monomer was cooled to 5°C, 9.3 g of a 2% by weight potassium persulfate aqueous solution was added as a polymerization initiator to prepare an aqueous monomer solution. In a 2-liter separable flask equipped with a stirrer and condenser, 1434 g of cyclohexane and 7.1 g of Rheodol SP-S10V (Kao Corporation, sorbitan monostearate) as a dispersant were placed. The mixture was heated to an internal temperature of 60°C using a water bath and stirred to dissolve the dispersant in the cyclohexane. After passing nitrogen through the solution in the separable flask to reduce the dissolved oxygen in the cyclohexane to 0.1 ppm or less, 350 g of monomer aqueous solution was added dropwise using a dropping funnel while stirring with the stirrer, and reverse-phase suspension polymerization was carried out at a polymerization temperature of 80°C. Subsequently, further monomer aqueous solution was added dropwise, and after the addition was complete, the mixture was heated for another 2 hours to complete the suspension polymerization, obtaining a spherical hydrated gel in cyclohexane. After stopping the rotation of the stirrer and allowing the generated hydrated gel to settle, the cyclohexane was removed by decantation, and the remaining hydrated gel was washed several times with cyclohexane to remove the dispersant adhering to the hydrated gel. The obtained spherical water-containing gel was spread on release paper and dried for 1 hour in a vacuum dryer at 130°C (vacuum level: 10,000-20,000 Pa) to obtain a polymer. The neutralization rate of this polymer was 72 mol%. Crosslinked polymer (A-1) was obtained by adjusting the particle size of this polymer to 150-300 μm using sieves with mesh sizes of 150 μm and 300 μm. The volume-average particle size of crosslinked polymer (A-1) was measured to be 220 μm using a particle analyzer (Retsch CAMSIZER XT). The water absorption ratio of physiological saline was 16 g / g.

[0077] <Manufacturing Example 2: Manufacturing of Crosslinked Polymer (A-2)> Cross-linked polymer (A-2) was obtained by following the same procedure as for the cross-linked polymer (A-1) in Production Example 1, except that the amount of ethylene glycol diglycidyl ether was changed to 1.5 g (1.3 wt% / acrylic acid). The volume-average particle size of the cross-linked polymer (A-2) was measured using a particle analyzer (Retsch CAMSIZER XT) and found to be 200 μm. The absorption ratio of physiological saline was 26 g / g.

[0078] <Manufacturing Example 3: Manufacturing of Crosslinked Polymer (B-1)> 50 g of cross-linked polymer (A-1) was mixed with 25 g of a 50 wt% potassium carbonate aqueous solution via a spray nozzle, and the mixture was heated at 130°C for 30 minutes. The mixture was then cooled to room temperature to obtain cross-linked polymer (B-1). The pH of the physiological saline solution of cross-linked polymer (B-1) was measured using the above method and found to be 7.4.

[0079] <Manufacturing Example 4: Manufacturing of Crosslinked Polymer (B-2)> The process was carried out in the same manner as in Production Example 3, except that the cross-linked polymer was changed from (A-1) to (A-2), and cross-linked polymer (B-2) was obtained. The pH of the physiological saline solution of cross-linked polymer (B-2) was measured using the method described above and was found to be 7.4.

[0080] <Manufacturing Example 5: Manufacturing of Crosslinked Polymer (C-1-a)> 10 g of cross-linked polymer (B-1) was mixed uniformly with 2.0 g of a 5 wt% hexadecyltrimethylammonium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ethanol solution by dropwise addition using a dropper. The mixture was then heated at 130°C for 30 minutes and cooled to room temperature to obtain cross-linked polymer (C-1-a). The volume-average particle size of the crosslinked polymer (C-1-a) was measured to be 220 μm using a particle analyzer (Retsch CAMSIZER XT). The absorption ratio of physiological saline was 13 g / g. The pH of the physiological saline solution was measured to be 7.4. Compound X in the crosslinked polymer in this example is hexadecyltrimethylammonium chloride.

[0081] <Manufacturing Example 6: Manufacturing of Crosslinked Polymer (C-1-b)> The method for producing the cross-linked polymer (C-1-b) was carried out in the same manner as in Production Example 5 for producing the cross-linked polymer (C-1-a), except that the ethanol solution of 5% by weight hexadecyltrimethylammonium chloride was replaced with 2 g of a cyclohexane solution of 5% by weight lecithin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from soybeans). The volume-average particle size of the cross-linked polymer (C-1-b) was measured to be 220 μm using a particle analyzer (Retsch CAMSIZER XT). The absorption ratio of physiological saline was 13 g / g. The pH of the physiological saline solution was measured to be 7.4. Compound X in the cross-linked polymer in this example is lecithin.

[0082] <Manufacturing Example 7: Manufacturing of Crosslinked Polymer (C-2-a)> The process was carried out in the same manner as in Production Example 5, except that the crosslinked polymer was changed from (B-1) to (B-2), and crosslinked polymer (C-2-a) was obtained. The volume-average particle size of the cross-linked polymer (C-2-a) was measured to be 200 μm using a particle analyzer (Retsch CAMSIZER XT). The absorption ratio of physiological saline was 22 g / g. The pH of the physiological saline solution was measured to be 7.3.

[0083] <Preparation of biological samples> Human-derived samples were obtained as urine, serum, and plasma (using K2EDTA as an anticoagulant). After obtaining these samples, 1 mL of each sample was centrifuged at 2,000 × g for 10 minutes, and the supernatant was collected after removing debris. For urine samples, 1 mL of the sample was used as is. For serum and plasma samples, 0.2 mL of the sample was mixed with 0.8 mL of phosphate-buffered saline (PBS) to create a 1 mL solution. The cell culture supernatant was prepared by subculturing HEK293 cells in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) by volume. Once the cells reached 80% confluence, the medium was replaced with FBS-free DMEM. After 48 hours of incubation, the conditioned medium was collected, and 1 mL of the medium was centrifuged at 2,000 × g for 10 minutes to remove debris. The supernatant was then used.

[0084] <Preparation of the crushed liquid> For use in the examples, RIPA Buffer [manufactured by Nacalai Tesque Co., Ltd.] and RNA purification lysis buffer (Buffer MLP, manufactured by MACHEREY-NAGEL) were prepared, and lysates 1 to 3 with the compositions shown in Table 1 were prepared. The types and contents of surfactants contained in RIPA Buffer are also shown. The notation in Table 1 is as follows: • RIPA Buffer: A surfactant-containing buffer manufactured by Nacalai Tesque Co., Ltd. • NP-40: Manufactured by Takara Bio Inc., Nonidet® P-40 (polyoxyethylene octylphenyl ether) Tween20: Polyoxyethylene (20) sorbitan monolaurate • CHAPS: Manufactured by Fujifilm Wako Pure Chemical Corporation, 3-[(3-collamidopropyl)dimethylammonio]propanesulfonate Tris: Tris buffer solution • PBS: Phosphate-buffered saline NaCl: Sodium chloride

[0085] [Table 1]

[0086] <Example 1> (1-1) Adsorption of EV onto crosslinked polymer [Step (1)] 55 mg of the cross-linked polymer (C-1-a) prepared in Production Example 5 was added to a spin column (BIORAD Co., Ltd., Microbio Spin Column), and then 1.0 mL of cell culture supernatant was added as a biological sample, and the mixture was allowed to stand at 25°C for 30 minutes. Subsequently, the spin column was spun down at 2000 × g for 60 seconds in a benchtop centrifuge (Chibitan-R) to remove unadsorbed solution. Then, 0.5 mL of physiological saline (0.9 wt%) was added, and the physiological saline was separated using the benchtop centrifuge (Chibitan-R). This washing procedure was repeated three times. This resulted in obtaining a polymer gel with adsorbed extracellular vesicles (EVs).

[0087] (1-2) Recovery of EV contents [Process (2)] To the polymer gel adsorbed with EV obtained in (1-1), 0.1 mL of RIPA Buffer (Nacalai Tesque) was added as a lysate, and the spin column was thoroughly mixed with a vortex mixer for 10 seconds, then allowed to stand for 30 minutes. After standing, the spin column was spun down in a benchtop centrifuge (Chibitan-R) at 2000 × g for 60 seconds to remove the gel and obtain the inclusion-containing liquid. The amount of CD9 recovered from the encapsulated liquid was measured using an ELISA kit (Hakarel, CD9 / CD9 ELISA kit for human exosome quantification), and the result was 4.0 ng. The amount of RNA recovered from the encapsulated solution was evaluated by diluting the solution with 0.3 mL of 1.0 mol / L guanidine thiocyanate solution, adding it to a NucleoSpin miRNA Column (MACHEREY-NAGEL), purifying the RNA according to the NucleoSpin miRNA Plasma (MACHEREY-NAGEL) protocol, and then measuring the RNA quantification using an RNA quantification kit (Promega, QuantiFluor RNA System). The measurement results for the amount of RNA recovered and the amount of RNA recovered in Comparative Example 1 (details described later) are shown in Table 2, with the amount of RNA recovered set to 100.

[0088] <Examples 2-4> In Example 1 (1-2), the same procedure as in Example 1 was performed except that the crushing solution was changed from RIPA Buffer to the crushing solutions listed in Table 2 to obtain the inclusion-containing liquid. The amount of CD9 recovered from the inclusion-containing liquid was measured using the same method as in Example 1, and the results were 0.6 ng, 1.4 ng, and 1.2 ng, respectively. The amount of RNA recovered from the encapsulated liquid was measured using the same method as in Example 1. The measurement results for the amount of RNA recovered and the amount of RNA recovered when the amount of RNA recovered in Comparative Example 1 is set to 100 are shown in Table 2.

[0089] <Example 5> In Example 1 (1-2), the same procedure as in Example 1 was performed except that the crushing liquid was changed from RIPA Buffer to Buffer MLP to obtain a liquid containing inclusions. The amount of RNA recovered from the encapsulated solution was measured by adding the solution to a NucleoSpin miRNA Column (MACHEREY-NAGEL), followed by RNA purification according to the protocol for NucleoSpin miRNA Plasma (MACHEREY-NAGEL), and then measuring the amount in the same manner as in Example 1. The measurement results of the RNA recovery amount and the RNA recovery amount when the RNA recovery amount of Comparative Example 1 is set to 100 are shown in Table 2.

[0090] <Comparative Example 1: Example where step (2) is not performed> In Example 1 (1-2), the procedure was the same as in Example 1, except that the lysate was changed from RIPA Buffer to saline solution (20% by weight sodium chloride, pH 7.0) which does not have lysating properties, in order to obtain an extracellular vesicle solution. The amount of CD9 recovered from the obtained extracellular vesicle solution was measured using the same method as in Example 1, and the result was 3.1 ng. The amount of RNA recovered was measured using the same method as in Example 1 after purifying the extracellular vesicle solution with an RNA purification kit (MACHEREY-NAGEL, NucleoSpin miRNA Plasma). The results of the RNA recovery measurements are shown in Table 2.

[0091] <Example 6> In Example 1 (1-1), the same procedure as in Example 1 was performed except that the biological sample was replaced with one of those listed in Table 2 (serum) to obtain the inclusion-containing solution. The amount of CD9 recovered from the obtained encapsulated liquid was 5.4 ng, as determined in the same manner as in Example 1. The amount of RNA recovered was measured using the same method as in Example 1. Table 2 shows the RNA recovery results and the RNA recovery amount when the RNA recovery amount in Comparative Example 2 (details described later) is set to 100.

[0092] <Example 7> In Example 1 (1-1), the biological sample was changed to one of those listed in Table 2 (serum), and in Example 1 (1-2), the lysate was changed from RIPA Buffer to Buffer MLP. The same procedure as in Example 1 was followed to obtain a solution containing inclusions. The amount of RNA recovered from the obtained encapsulated liquid was measured using the same method as in Example 1. The measurement results of the RNA recovery amount and the RNA recovery amount when the RNA recovery amount of Comparative Example 2 is set to 100 are shown in Table 2.

[0093] <Comparative Example 2: Example where step (2) is not performed> In Example 1 (1-1), the biological sample was changed to one of those listed in Table 2 (serum), and in Example 1 (1-2), the lysate was changed from RIPA Buffer to saline solution (20% by weight sodium chloride, pH 7.0). The same procedure as in Example 1 was followed to obtain a solution containing the inclusions. The amount of CD9 recovered from the obtained encapsulated liquid was measured using the same method as in Example 1, and was found to be 4.4 ng. The amount of RNA recovered was measured using the same method as in Example 1 after purifying the encapsulated liquid with an RNA purification kit (MACHEREY-NAGEL, NucleoSpin miRNA Plasma). The results of the RNA recovery measurements are shown in Table 2.

[0094] <Example 8> In Example 1 (1-1), the same procedure as in Example 1 was performed except that the biological sample was changed to one of those listed in Table 3 (urine) to obtain a solution containing the encapsulated substance. The amount of CD9 recovered from the obtained encapsulated liquid was 3.8 ng, as determined in the same manner as in Example 1. The amount of RNA recovered was measured using the same method as in Example 1. Table 3 shows the RNA recovery results and the RNA recovery amount when the RNA recovery amount in Comparative Example 3 (details described later) is set to 100.

[0095] <Examples 9-10> In Example 1 (1-1), the same procedure as in Example 1 was performed to obtain a liquid containing encapsulated material, except that the cross-linked polymer particles were changed to those listed in Table 3 and the biological sample was replaced with the one listed in Table 3 (urine). The amount of CD9 recovered from the obtained encapsulated liquid was 3.1 ng and 3.4 ng, respectively, as determined in the same manner as in Example 1. The amount of RNA recovered was measured using the same method as in Example 1. Table 3 shows the results of the RNA recovery measurements and the amount of RNA recovered when the amount of RNA recovered in Comparative Example 3 (details of which will be described later) is set to 100.

[0096] <Example 11> In Example 1 (1-1), the biological sample was changed to one of those listed in Table 3 (urine), and in Example 1 (1-2), the lysate was changed from RIPA Buffer to Buffer MLP. The same procedure as in Example 1 was followed to obtain a solution containing the inclusions. The amount of RNA recovered from the obtained encapsulated liquid was measured using the same method as in Example 1. Table 3 shows the measurement results of the RNA recovery amount and the RNA recovery amount when the RNA recovery amount of Comparative Example 3 (details described later) is set to 100.

[0097] <Comparative Example 3> In Example 1 (1-1), the biological sample was changed to one listed in Table 3 (urine), and the cross-linked polymer particles were replaced with those listed in Table 3. In Example 1 (1-2), the lysate was changed from RIPA Buffer to saline solution (20% by weight sodium chloride, pH 7.0). The same procedure as in Example 1 was followed to obtain a solution containing the encapsulated material. The amount of CD9 recovered from the obtained encapsulated liquid was measured in the same manner as in Example 1, and was found to be 2.1 ng. The amount of RNA recovered was measured using the same method as in Comparative Example 1. The results of the RNA recovery measurements are shown in Table 3.

[0098] <Example 12> In Example 1 (1-1), the biological sample was changed to one of those listed in Table 3 (plasma), and in Example 1 (1-2), the lysate was changed from RIPA Buffer to Buffer MLP. The same procedure as in Example 1 was followed to obtain the inclusion-containing solution. The amount of RNA recovered from the obtained encapsulated liquid was measured using the same method as in Example 1. The results of the RNA recovery measurement are shown in Table 3.

[0099] [Table 2]

[0100] [Table 3]

[0101] As shown in Tables 2 and 3, when comparing cases with the same bio-derived material but different recovery methods (for example, Examples 1-5 and Comparative Example 1, Examples 6-7 and Comparative Example 2, and Examples 8-11 and Comparative Example 3), the RNA recovery amount was significantly higher in the example methods than in the comparative example methods. From these results, it can be seen that the present invention provides a method for recovering EV encapsulated materials at high concentration and high yield.

Claims

1. A method for recovering contents from an extracellular vesicle, comprising steps (1) and (2), The above step (1) is a step of contacting a biological sample with a crosslinked polymer to obtain a polymer gel on which extracellular vesicles are adsorbed. The above step (2) is a step of disrupting the extracellular vesicles adsorbed onto the polymer gel and recovering the contents of the extracellular vesicles. A method for recovering contents from extracellular vesicles, wherein step (2) includes a step (2A) of disrupting extracellular vesicles adsorbed on a polymer gel using a disruption solution containing a surfactant, and / or a step (2B) of disrupting extracellular vesicles adsorbed on a polymer gel by ultrasonic vibration.

2. The method according to claim 1, wherein the crosslinked polymer is a crosslinked polymer comprising monomer units having an acidic group and / or a neutralizing base thereof, and contains a compound having at least one group selected from the group consisting of a cationic group and a hydroxyl group.

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