027 – Isolation of exosomes from human plasma: comparison of size exclusion chromatography columns with ultracentrifugation.
Stefano Gelibter (1) – Giulia Marostica (1) – Lorena Fabbella (1) – Tommaso Croese (1) – Mario Orrico (1) – Annamaria Finardi (1) – Roberto Furlan (1)
San Raffaele Hospital, Clinical neuroimmunology Unit,Institute of Experimental Neurology (INSPE), MILANO, Italy (1)
Exosomes can be detected in plasma and other biological fluids, containing material of the origin cells. Importantly, they have a composition reflecting the state of cell activation, are influenced by pathologic processes and are known to cross blood-brain-barrier.
Our aim is to compare plasma exosomes isolation and disruption methods in order to choose the best one for further protein content analysis and to investigate the effect of -80 ¡C storage on exosomes concentration.
Blood samples has been collected from healthy controls(n=5). We compared two different methods of exosomes isolation: ultracentrifugation (UC) (100000 g x 90 minutes twice, 4¡C) vs size exclusion cromatography (SEC ÐqEV commercial kitÐIzon). To compare SEC and UC, concentration and size distribution of particles has been assessed with tunable resistive pulse sensing (TRPS Ð qNano, Izon), along with total protein concentration (Pierce BCA protein assay kit) to obtain particle-to-protein ratio, a measure of particle purity. Concentration has been again assessed after 4 week of storage at -80¡C. In addition to this, transmission electron microscopy (TEM) has been performed.
Probe sonication (Branson digital sonifier) has been compared to osmotic shock (dilution 1:10 in distilled water) to choose the best exosome disruption method.
Firstly, we found that plasma particles size distribution and concentration did not differ between UC and SEC and both methods isolated particles with a size range equivalent to exosomes. No significant differences were found between qEV and UC in the number of vesicles isolated from the same plasma volume, even if SEC allowed to isolate a higher number. qEV columns outperformed UC in sample purity, as demonstrated with the significative difference of particle/protein ratio (p=0.01) and confirmed with TEM.
Probe sonication was significantly better in exosome disruption (average reduction of concentration 91% vs 72%, p <0.03).
In -80¡C stored samples, exosomes concentration significantly decreased (p=0.01), with an average reduction of 62%.
Our data suggest that SEC outperforms UC in sample purity for plasma exosome isolation. In addition to this, we identified probe sonication as a valid method for exosome disruption for further analysis.
Our results could allow us to isolate plasma exosome in patients suffering from neurological diseases helping to identify new biomarkers which can be relatively easily detected in a non invasive way in clinical practice.