Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents

TLDR

Extracellular vesicles (EVs) are a rapidly growing research area, yet no consensus exists on their isolation, with ultracentrifugation being standard but newer rapid methods such as size‑exclusion chromatography (SEC), polyethylene glycol (PEG) precipitation, and protein‑organic solvent precipitation (PROSPR) emerging. The study aimed to assess how these isolation methods affect the characteristics of plasma‑derived EVs. Plasma EVs were isolated by SEC, PEG, and PROSPR, then compared for total protein, nanoparticle tracking analysis, cryo‑electron microscopy, and EV‑marker expression, and their impact on recipient cell viability was also evaluated. SEC yielded the lowest protein contamination, preserved EV markers, and maintained cell viability, whereas PEG and PROSPR retained more soluble proteins, failed to detect key markers, and reduced recipient cell viability, underscoring the importance of method selection for downstream applications.

Abstract

Abstract Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR and their total protein content, NTA and Cryo-electron microscopy profiles and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro . These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs.