Choice of size-exclusion chromatography column affects recovery, purity, and miRNA cargo analysis of extracellular vesicles from human plasma

Aim: The miRNA cargo of plasma extracellular vesicles (EVs) is commonly studied for its biomarker potential. However, isolation of EVs from human plasma is challenging. Although size-exclusion chromatography (SEC) is commonly used to isolate plasma EVs, SEC does not completely separate EVs from other miRNA carriers such as cells, lipoproteins, and proteins. Recently, new SEC columns were introduced, but hitherto, no systematic study was performed to compare the recovery and purity of plasma EVs using both traditional and new columns. In this study, we investigated the recovery of EVs and separation efficacy from lipoproteins and proteins using different SEC columns, and how recovery and separation affect miRNA cargo analysis. Methods: EVs were isolated from pooled (n = 5) platelet-depleted plasma using 10 different SEC columns. For each column, three EV-enriched fractions were pooled and concentrations of EVs, lipoproteins, proteins, and miRNAs were measured by flow cytometry, enzyme-linked immunosorbent assay (ELISA), Bradford assay, and qRT-PCR, respectively. Results: Our results show that the resin pore size affects all measured parameters: a small pore size increases recovery of EVs and quantity of miRNA, but decreases the separation efficacy compared to a large pore size. Regression analysis showed that the investigated miRNAs are more strongly associated with EVs than with lipoproteins or proteins. Conclusion: The choice of a SEC column markedly affects the recovery, separation efficacy, and miRNA cargo analysis of human plasma-derived EVs. We recommend either using SEC columns with a 70-nm pore size due to their superior EV purity or studying the effect of non-EV particles on the miRNAs of interest.