Molecular evaluation of different enrichment methods of extracellular vesicles from bio-banked serum from human healthy subjects
Extracellular vesicles (EVs) from human body fluids are important tools for biomarker discovery and for exploring the mechanisms underlying different pathologies, including infectious diseases. The translation of EVs research into clinical practice is however hindered by the variability in EVs pre-clinical investigations, thus standardisation of analytical procedures and reporting policies is essential. Human serum is a key biological matrix for biomarker discovery and commonly stored within biobanks. Here we evaluated the impact of different enrichment strategies on EVs downstream analyses. EVs were enriched from 250µl of bio-banked serum using ultracentrifugation (UC), size exclusion chromatography-based methods (ExoSpin - ES, qEV1-35nm – i35 and qEV1-70nm – i70) and ExoRNeasy (ER), prior to morphological evaluation, enumeration, surface phenotyping and multi-'omics characterisation.  All methods enriched adequate amounts of small EVs bearing on their surface tetraspanins, although at different concentrations. The most efficient method for proteomics analyses was qEV1-70nm, followed by ES, which however was more susceptible to contamination from serum proteins. EV-miRNA cargo was efficiently characterised in UC-, ES- and ER-EVs, with the latter providing the highest coverage of vesicular miRNA. Our results support the possibility of using bio-banked serum for EV-based research and further highlight the importance of selecting EV enrichment methods, since they shapes both miRNA and protein landscape.
Extracellular vesicles (EVs) from human body fluids are important tools for biomarker discovery and for exploring the mechanisms underlying different pathologies, including infectious diseases. The translation of EVs research into clinical practice is however hindered by the variability in EVs pre-clinical investigations, thus standardisation of analytical procedures and reporting policies is essential. Human serum is a key biological matrix for biomarker discovery and commonly stored within biobanks. Here we evaluated the impact of different enrichment strategies on EVs downstream analyses. EVs were enriched from 250µl of bio-banked serum using ultracentrifugation (UC), size exclusion chromatography-based methods (ExoSpin - ES, qEV1-35nm – i35 and qEV1-70nm – i70) and ExoRNeasy (ER), prior to morphological evaluation, enumeration, surface phenotyping and multi-'omics characterisation.  All methods enriched adequate amounts of small EVs bearing on their surface tetraspanins, although at different concentrations. The most efficient method for proteomics analyses was qEV1-70nm, followed by ES, which however was more susceptible to contamination from serum proteins. EV-miRNA cargo was efficiently characterised in UC-, ES- and ER-EVs, with the latter providing the highest coverage of vesicular miRNA. Our results support the possibility of using bio-banked serum for EV-based research and further highlight the importance of selecting EV enrichment methods, since they shapes both miRNA and protein landscape.
IRCCS Sacro Cuore Don Calabria Hospital