KCS

Extracellular vesicles (EVs) are cell-derived membrane-covered particles containing proteins, lipids, nucleic acids and other biological molecules. EVs play an essential role in the communication between cells via transporting pathologically important molecules. As the pathological importance of EVs increases, the expectation of EVs as a biomarker is growing. Subpopulations of EVs are divided into exosomes (30-100 nm in diameter) and microvesicles (100-1000 nm) according to their sizes and therefore, it is important to separate them because they differ in cellular origins, contents, and lipid compositions. However, it is challenging to isolate EVs while each of the separation methods such as ultracentrifugation, size exclusion chromatography, filtration, and precipitation has its own limitation. Field-flow fractionation (FFF) is a separation method capable of fractionating particles from nano to micron size in aqueous solution without using packing materials. In this study, exosomes and microvesicles secreted from cells were separated by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) with field programming and monitored by UV and multi-angle light scattering (MALS) detectors. Eluted exosomes and microvesicles were identified by western blotting and their sizes were determined by MALS and dynamic light scattering (DLS). Further, lipidomic analysis was performed by nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry to investigate the difference in lipid compositions between exosomes and microvesicles.