Purification of extracellular vesicles by size-exclusion chromatography using cross-linked agarose gels: the role of pore size in lipoprotein contamination

Extracellular vesicles (EVs) are pivotal in intercellular communication and hold promise for diagnostic and therapeutic applications. Their isolation from complex biological fluids like plasma, however, poses significant challenges. This study examined the impact of pore size in agarose gels on the yield and purity of EVs isolated by Size-Exclusion Chromatography (SEC). We systematically compared the performance of agarose gels with different degrees of crosslinking (Sepharose CL-2B, CL-4B, and CL-6B) using both HPLC-SEC and gravity-based protocols, utilizing the supernatant from isolated human platelets redispersed in an additive solution containing plasma as the source of EVs. The experimental design involved the precise determination of EV elution volumes, identification of EV fractions based on associated markers, and assessment of co-eluted plasma constituents. The findings indicated that smaller pore sizes of the gels led to increased co-elution of lipoproteins with EVs. Yet, they also enhanced EV yields, highlighting a trade-off between yield and purity. Specifically, the CL-6B gel, with the smallest pore size, demonstrated the highest EV yield and lipoprotein contamination among the compared gels. Consequently, the selection of SEC gel for EV purification should be tailored to the requirements of the subsequent analysis, balancing yield and purity considerations.