Method for recovering extracellular vesicles

Abstract

The present invention provides recovering an extracellular vesicle(s) having high purity from an extracellular vesicle-containing sample. Specifically, the present invention provides a method of recovering an extracellular vesicle(s), comprising: (1) treating an extracellular vesicle-containing sample with a chelating agent; and (2) separating the extracellular vesicle(s) from the extracellular vesicle-containing sample treated with the chelating agent.

1 : A method of recovering extracellular vesicles, the method comprising:
 (1) adding a chelating agent to a sample comprising extracellular vesicles, thereby obtaining a solution comprising the chelating agent and the extracellular vesicles;   (2) treating the sample comprising extracellular vesicles with a chelating agent in the solution; and   (3) separating the solution obtained in (2) into (i) a liquid portion comprising the chelating agent and (ii) a precipitation portion comprising the extracellular vesicles,   (4) removing the liquid portion comprising the chelating agent; and   (5) recovering the precipitation portion comprising the extracellular vesicles.   
     
     
         2 : The method of  claim 1 , wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), glycoletherdiaminetetraacetic acid (EGTA), hydroxyethyl ethylenediaminetriacetic acid (HEDTA), hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediaminetetra (methylenephosphonic acid) (EDTMP), a salt thereof, and a mixture thereof. 
     
     
         3 : The method of  claim 1 , wherein the extracellular vesicle-comprising sample is treated with 1 to 200 mM of the chelating agent. 
     
     
         4 : The method of  claim 3 , wherein the extracellular vesicle-comprising sample is treated with at least 40 mM of the chelating agent. 
     
     
         5 : The method of  claim 1 , wherein the extracellular vesicle(s) is an exosome. 
     
     
         6 : The method of  claim 1 , wherein the separating is performed by a precipitation method using an extracellular vesicle membrane-binding substance or an ultracentrifugation method of the extracellular vesicle-comprising sample. 
     
     
         7 : The method of  claim 6 , wherein the extracellular vesicle membrane-binding substance is an antibody against CD9. 
     
     
         8 : The method of  claim 1 , wherein the extracellular vesicle-comprising sample is a body fluid or a culture supernatant. 
     
     
         9 : The method of  claim 1 , wherein the extracellular vesicle-comprising sample is a blood sample, urine, or a breast fluid. 
     
     
         10 : A method of analyzing an extracellular vesicle(s), the method comprising:
 (1) recovering extracellular vesicles by the method of  claim 1 , wherein the extracellular vesicles comprise a component to be analyzed;   and   (2) analyzing the recovered extracellular vesicles,   (i) wherein w % ben the recovered extracellular vesicles comprise the component to be analyzed, the component in the recovered extracellular vesicles is analyzed by at least one method selected from the group consisting of an immunoassay, hybridization method, gene amplification method, mass spectrometry, proteome analysis and metabolome analysis, or   (ii) the recovered extracellular vesicles are analyzed with at least one instrument selected from the group consisting of a particle analytical instrument, an electron microscope and a flow cytometer.   
     
     
         11 : The method of  claim 10 , wherein the recovered extracellular vesicles comprise a protein, and the protein in the recovered extracellular vesicles is analyzed by at least one method selected from the group consisting of an immunoassay, mass spectrometry and proteome analysis. 
     
     
         12 - 20 : (canceled) 
     
     
         21 : The method of  claim 1 , wherein the sample is treated with the chelating agent for at least one minute. 
     
     
         22 : The method of  claim 1 , wherein the treating is performed at a temperature of from 15 to 30° C. 
     
     
         23 : The method of  claim 1 , wherein the sample comprising extracellular vesicles is treated with EDTA. 
     
     
         24 : The method of  claim 1 , wherein the sample comprising extracellular vesicles is treated with EGTA. 
     
     
         25 : The method of  claim 1 , further comprising preparing the sample comprising extracellular vesicles before (1), wherein the sample comprising extracellular vesicles is plasma. 
     
     
         26 : The method of  claim 1 , wherein the sample comprising extracellular vesicles comprises a sample other than urine. 
     
     
         27 : The method of  claim 6 , wherein the extracellular vesicle membrane-binding substance is bound to a solid phase. 
     
     
         28 : The method of  claim 6 , wherein the extracellular vesicle membrane-binding substance is bound to magnetic beads. 
     
     
         29 : The method of  claim 1 , further comprising suspending the recovered precipitation portion in a solution which does not comprise a chelating agent, thereby obtaining a suspension which comprises the extracellular vesicles and which does not comprise a chelating agent.