JP-2026075615-A - Method for recovering contents from extracellular vesicles

Abstract

[Problem] To provide 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 (2A) of disrupting the extracellular vesicles adsorbed on the polymer gel using a disruption solution containing a surfactant and/or a step (2B) of disrupting the extracellular vesicles adsorbed on the polymer gel by ultrasonic vibration. [Selection Diagram] None

Inventors

• 梶原 竜太
• 鈴木 涼介

Assignees

• 三洋化成工業株式会社

Dates

Publication Date

20260508

Application Date

20251021

Priority Date

20241022

Claims (2)

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.

Description

This invention relates to a method for recovering contents from extracellular vesicles. Extracellular vesicles (hereinafter also called "EVs") secreted from 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 originate, and are useful as biomarkers for evaluating various diseases. To evaluate diseases and other conditions using EV inclusions as biomarkers, a technology is needed to recover the inclusions at high concentration and high yield. Examples of such technologies are known, such as those described in Patent Documents 1 and 2. International Publication No. 2022/211006International Publication No. 2020/090859 The present invention provides a method for recovering contents from extracellular vesicles, comprising steps (1) and (2). Step (1) involves contacting a biological sample with a cross-linked polymer to obtain a polymer gel on which extracellular vesicles are adsorbed, and step (2) involves disrupting the extracellular vesicles adsorbed on the polymer gel and recovering the contents of the extracellular vesicles. In the present invention, 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. <Step (1)> Step (1) is a step in which a biological sample is brought into contact with a cross-linked polymer to obtain a polymer gel on which extracellular vesicles are adsorbed. <Cross-linked polymer> Examples of crosslinked polymers used in step (1) include crosslinked polymers containing monomer units having an acidic group and/or a neutralizing base thereof. In this specification, "acidic group" refers to a group having a dissociable proton, and includes, but is not limited to, a carboxylic acid group (-COOH), a sulfonic acid group [-S(=O) ₂ (OH)], a sulfate group [-O-S(=O) ₂ (OH)], a phosphonic acid group [-P(=O)(OH) ₂ ], and a phosphate group [-O-P(=O)(OH) ₂ ]. In this specification, "neutralizing base of an acidic group" means a neutralizing salt with any cation. Specific examples of cations include metals (such as monovalent or divalent metals), and cations represented by the formula: NR₄⁺ (wherein each R independently represents a hydrogen atom or an optionally substituted hydrocarbon group, and two or three Rs may form a ring with adjacent nitrogen atoms). Examples of monovalent metals include alkali metals such as lithium, sodium, and potassium. Examples of divalent metals include alkaline earth metals such as magnesium, calcium, and barium, as well as lead, zinc, and tin. In the cation represented by the formula NR 4 + , if R is an optionally substituted hydrocarbon group, the hydrocarbon group may be, for example, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. Examples of alkyl groups include C1-6 alkyl groups such as methyl group, ethyl group, propyl group (n-propyl group or isopropyl group), and butyl group (n-butyl group, isobutyl group, sec-butyl group, or t-butyl group). Examples of cycloalkyl groups include C5-14 cycloalkyl groups such as cyclopentyl group and cyclohexyl group. Examples of aryl groups include C6-14 aryl groups such as phenyl group and naphthyl group. Examples of aralkyl groups include C7-14 aralkyl groups such as benzyl group and phenethyl group. Examples of substituents that can be substituted on the hydrocarbon group include halogen atoms, hydroxyl groups, and mercapto groups. In the cation represented by the formula NR4 + , if two or three R atoms form a ring with adjacent nitrogen atoms, the ring may be a monoring such as a pyridine ring or an imidazole ring, or a fused ring such as a quinoline ring. The ring may have one or more substituents, such as halogen atoms, hydroxyl groups, amino groups, alkyl groups, haloalkyl groups, hydroxyalkyl groups, and N,N-dialkylamino groups. The number of substituents may be, for example, one, two, or three. 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 acrylic acid, methacrylic acid, and crotonic acid, which are unsaturated monocarboxylic acids having 3 to 10 carbon atoms. Examples of unsaturated dicarboxylic acids (including anhydrides in this specification) include maleic acid, fumaric acid, citraconic acid, itaconic acid, and their anhydrides. 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-sulfoet