Isolation and Characterization of Microvesicles in Blood Plasma by Size-exclusion Chromatography

Extracellular vesicles (EVs) (50 nm to 2 μm) are a heterogeneous collection of membrane-enclosed structures that are located in the extracellular space and released from the surface of many different cell types into different bodily fluids. EVs includes exosomes (30-100nm), apoptotic bodies (\u3e1000 nm), and microvesicles (100 nm-1000nm) which may deliver bioactive cargo such as lipids, signaling molecules, growth factors, mRNA and non-coding mRNA that are released from the cell of origin to their target cells. Recent studies demonstrate that plasma-derived microvesicles may play a role in cell-cell communication, may potentially be diagnostic, or are even prognostic biomarkers of diseases. However, to establish any of these roles for MVs, we need to be able to isolate them from blood plasma easily without contamination by proteins and lipids that are quite abundant. Therefore, isolating extracellular vesicles from blood will be of great importance in understanding their biological role and to use EVs as biomarkers of disease. In this study, we built size exclusion chromatography columns and then used them to isolate MVs from platelets-free blood plasma samples and assessed their effectiveness compared to typical centrifugation protocols. We measured the protein concentrations of each eluted fractions and found that fractions 19-26 had a higher protein concentration and, as expected, protein content gradually increased with each fraction. Using flow cytometry, we enumerated CD41+ (platelet) and CD62e+ (endothelial) derived MVs. Unlike protein, fractions 9-12 contained the highest concentrations of the platelet-derived vesicles similar to previous studies. In conclusion, our custom-made size exclusion chromatography column performed as expected and separated blood plasma MVs from proteins effectively