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 × 106vs 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 × 1010vs 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.