Method for isolating extracellular vesicles using cations
The present invention relates to a method for isolating extracellular vesicles using cations, and more particularly, to a method for isolating extracellular vesicles from various samples by using the affinity between the extracellular vesicles and cations. A method for isolating extracellular vesicles according to the present invention does not require expensive equipment, can be applied irrespective of sample amount, and has the advantage of being capable of efficiently isolating the extracellular vesicles while preserving the shape or characteristics thereof. Moreover, the method according to the present invention can be combined with existing isolation methods to maximize extracellular vesicle isolation efficiency, and can be applied to disease diagnosis, disease treatment, and multi-omics research using isolated extracellular vesicles, as well as to research on the properties of extracellular vesicles.
1. A method for isolating extracellular vesicles, the method comprising:
(a) adding cations to a biological sample containing extracellular vesicles at a concentration sufficient to form an extracellular vesicle-cation insoluble complex, and wherein the cations have a specific affinity for the extracellular vesicles, with the proviso that if the cations are calcium ions, then the calcium ions are added at a concentration of 10 mM or greater, wherein the cations exclude sodium, lithium, or potassium ions;
(b) inducing a specific binding between the extracellular vesicles and the cations by reacting the cations with the extracellular vesicles contained in the biological sample, thereby forming an extracellular vesicle-cation insoluble complex;
(c) separating the extracellular vesicle-cation insoluble complex from the biological sample, wherein the separating is performed by using at least one method selected from the group consisting of centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, density gradient ultracentrifugation, precipitation, polymer-based precipitation, and organic solvent precipitation; and
(d) separating the cations from the complex to purify the extracellular vesicles.
2. The method of claim 1 , wherein the biological sample is at least one sample selected from the group consisting of mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, a tissue extract, a cancer tissue, serum, blood plasma, saliva, tears, sweat, urine, feces, cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil, and a fermented food.
3. The method of claim 1 , wherein the cations are metal cations.
4. The method of claim 1 , wherein step (d) comprises at least one method selected from the group consisting of: adding a chelating agent to the separated extracellular vesicle-cation complex; changing a pH value; and changing the concentration of at least one element selected from the group consisting of imidazole, histidine, ethylenediamine tetraacetate acid (EDTA), and a salt.
5. The method of claim 4 , wherein the chelating agent is at least one chemical selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris-(carboxymethyl)ethylenediamine (TED), ethylenediamine, ethylenediamine tetraacetate acid (EDTA), alkylene diamine triacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylene glycol-bis((3-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), phosphoserine, and 1,4,7-triazocyclononane (TACN).
6. The method of claim 1 , further comprising pre-treating the sample before step (a).
7. The method of claim 1 , further comprising post-treating the purified extracellular vesicles after step (d).
8. The method of claim 1 , further comprising adding a polymer or salting-out ions in step (a).
9. The method of claim 8 , wherein the polymer or salting-out ions are added before, simultaneously with, or after the addition of the cations.
10. The method of claim 8 , wherein the polymer is polyethylene glycol (PEG) or polyoxazoline.
11. The method of claim 10 , wherein the polyoxazoline is poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), or poly(2-propyl-2-oxazoline) (PPOZ).