Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy

Milica Popović, Ario de Marco


Extracellular vesicles (EVs) play a pivotal role in cell to cell signalling in both physiological and pathological conditions. Based on their biogenesis, three main classes of EVs are recognized: exosomes, microvesicles and apoptotic bodies. Exosomes are cell-derived vesicles (EVs) present in many body fluids (blood, urine, milk, cerebrospinal fluid) ranging in size from 30 to 150 nm. Due to their involvement in numerous physiological and pathological events, cell derived exosomes in bodily fluids represent a unique source of clinically relevant and non-invasive biomarkers. Since biomolecule content present in exosomes reflects the state of the parent cell, exosome analysis and characterization may provide valuable information about the presence of aberrant processes in the cells from which they originated. Because of the large and heterogeneous scientific community working with exosomes, several purification strategies have been applied so far, which yield EV fractions largely differing for quantity and quality. Most of the present exosome isolation approaches based on ultracentrifugation (UC), ultrafiltration (UF) or precipitation, are inefficient and hard to standardize, thereby creating low reproducibility in sample quality and potentially misleading results because highly sensitivity downstream analytical techniques, such as mass spectrometry, can detect even minute traces of co-isolated contaminants. Furthermore, loss of certain exosomal fractions during purification process or damage of exosomal membrane integrity can also alter final protein and RNA profiles. As a consequence, there is a strong interest in consensus principles for the exosome purification and the search for reliable methods for selective isolation of EV sub-populations. In the present manuscript, we critically overview the most commonly used techniques used for exosome preparation such as ultra-centrifugation, size-based isolation methods, precipitation and immunoaffinity (IA) and their respective applicability for purification of exosomes from clinically relevant samples.