Comparison of extracellular vesicle isolation processes for therapeutic applications.

While extracellular vesicles (EVs) continue to gain interest for therapeutic applications, their clinical translation is limited by a lack of optimal isolation methods. We sought to determine how universally applied isolation methods impact EV purity and yield. EVs were isolated by ultracentrifugation (UC), polyethylene glycol precipitation, Total Exosome Isolation Reagent, an aqueous two-phase system with and without repeat washes or size exclusion chromatography (SEC). EV-like particles could be detected for all isolation methods but varied in their purity and relative expression of surface markers (Alix, Annexin A2, CD9, CD63 and CD81). Assessments of sample purity were dependent on the specificity of characterisation method applied, with total particle counts and particle to protein (PtP) ratios often not aligning with quantitative measures of tetraspanin surface markers obtained using high-resolution nano-flow cytometry. While SEC resulted in the isolation of fewer particles with a relatively low PtP ratio (1.12 × 10⁷ ± 1.43 × 10⁶ vs highest recorded; ATPS/R 2.01 × 10⁸ ± 1.15 × 10⁹, p ⩽ 0.05), EVs isolated using this method displayed a comparatively high level of tetraspanin positivity (e.g. ExoELISA CD63⁺ particles; 1.36 × 10¹¹ ± 1.18 × 10¹⁰ vs ATPS/R 2.58 × 10¹⁰ ± 1.92 × 10⁹, p ⩽ 0.001). Results originating from an accompanying survey designed to evaluate pragmatic considerations surrounding method implementation (e.g. scalability and cost) identified that SEC and UC were favoured for overall efficiency. However, reservations were highlighted in the scalability of these methods, which could potentially hinder downstream therapeutic applications. In conclusion, variations in sample purity and yield were evident between isolation methods, while standard non-specific assessments of sample purity did not align with advanced quantitative high-resolution analysis of EV surface markers. Reproducible and specific assessments of EV purity will be critical for informing therapeutic studies.