Enhancing extracellular vesicles purity from plasma: A comparative study of isolation strategies.

Extracellular vesicles (EVs) are nanosized, membrane-bound particles found in various body fluids including plasma, saliva, urine which are released by cells under physiological and pathological conditions. They serve as promising biomarkers due to their cargo of nucleic acids, proteins and lipids reflecting the state of their cells of origin. However, isolating EVs from plasma remains technically challenging because of the high abundance of lipoproteins and soluble proteins with overlapping size and density characteristics. While individual isolation techniques such as ultracentrifugation (UC), size exclusion chromatography (SEC) and precipitation (Precip) have been widely employed, none can fully eliminate non-EV contaminants. In this study, we evaluated two combined isolation approaches - UC followed by Precip and SEC followed by Precip - to improve the yield and purity of plasma-derived EVs. Comprehensive characterization of EVs included assessment of protein and lipid content, particle size distribution and purity. Our results showed that combining SEC with a precipitation reagent, particularly ExoQuick-TC, maintained EVs integrity and sample purity. In contrast, combining UC with Precip resulted in lower EVs recovery and required larger plasma volumes, limiting its applicability in clinical context. Overall, our study suggests that SEC followed by Precip is a suitable strategy for obtaining EV-enriched plasma samples compatible with downstream characterization and biomarker studies.