Enrichment of Immune Cell‐Derived Extracellular Vesicles From Plasma Using 35 and 70 nm Size‐Exclusion Chromatography Columns of Different Sizes

Extracellular vesicles (EVs) from blood plasma are promising biomarkers, as they carry surface markers indicative of their cell of origin. Size-exclusion chromatography (SEC) is commonly employed for EV enrichment, but the choice of pore size and plasma volume can significantly impact the yield, purity, and composition of isolated EVs. In this study, we systematically compared Izon SEC columns with pore sizes of 35 and 70 nm, using either 500 µL plasma (qEVoriginal, "small" column) or 10 mL plasma (qEV10, "large" column). Due to limited material obtained from small columns, fractions had to be pooled for downstream analyses, precluding detailed characterization of individual fractions. In contrast, the larger columns provided sufficient material to analyse each fraction separately, across multiple platforms, including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), single-EV flow cytometry, MACSPlex surface protein array, immunoblotting, and LC-MS/MS. These analyses consistently identified fractions 1-3 as "EV-rich," characterized by enrichment of EV markers and reduced levels of abundant plasma proteins. Moreover, a comparison of pore sizes demonstrated that the 70 nm column yielded a higher EV recovery with improved purity compared to the 35 nm column, including a greater abundance of immune cell-derived markers. Together, these findings established that the large 70 nm SEC columns are optimal for isolating EV-rich fractions from plasma, maximizing both EV yield and purity, while minimizing non-EV contaminants.