Isolation and Characterization of Human Blood Extracellular Vesicles: a Promising Method of Fluid Biopsy

Extracellular vesicles (EV) are nanoscale vesicles that were previously thought to be secreted into the bloodstream by apoptotic cells. Today, EVs have been demonstrated to be secreted by almost all cells in the body, which contain valuable biomarkers for early diagnosis of a wide variety of diseases, particularly cancers. Efficient separation of EVs encounter challenges due to the presence of proteins and lipoproteins. Hence, the need for a time-saving and non-invasive diagnostic method with the ability to quantify isolated EVs, which is also applicable to resource-limited situations, can be the major necessities to advance EV-based diagnostic studies. The aim of the present study was to provide a relatively rapid and efficient method for isolating EV from blood as an important biological fluid in humans.

In this case study, with the combination of size exclusion chromatography and ultracentrifuge, EVs were successfully isolated from human blood plasma samples within an hour. To confirm the isolation process according to MISEV guidelines, three tetraspanin membrane proteins CD63, CD81, CD9, and one luminal protein TG101, were evaluated as positive protein markers and connexin protein as a negative marker by Western blot.

The maximum presence of positive markers and the absence of negative markers in F7 to F9 fractions were confirmed. Additionally, their size distribution evaluation was done using the DLS technique with an average diameter in the range of 100 nm. Quantification of the protein content of EVs was also performed by BCA assay.

The present study shows that the combination of high-speed centrifugation and size-based chromatography methods is very effective in isolating EV from the blood plasma of individuals