Extracellular vesicles (EVs) are membranous vesicles released by most cell types and play a pivotal role in cell-cell communication. Based primarily on their size (diameter) and presumed biogenesis pathways, EVs can be broadly classified into three main classes: exosomes (40-150 nm diameter), shed microvesicles (also referred to as oncosomes, 50-1,000 nm diameter), and apoptotic bodies (500-5,000 nm diameter). Previously, we compared traditional methods for isolating and characterising exosomes released into cell culture medium using the colorectal cancer cell line LIM1863 as a model [Tauro et al., Methods PMID: 22285593]. Three exosome isolation methods - ultracentrifugation (UC-Exos), OptiPrepTM density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos) – were evaluated using MS-based proteomics and label-free peptide spectral counting was employed to determine relative exosome enrichment. Our findings revealed that IAC is the preferred method for enriching exosomes from cell culture media.
Due to a considerable overlap in size (diameters) and buoyant densities of different EV types, along with a lack of specific vesicle surface makers, a major ongoing challenge in the EV field is to establish orthogonal methods that allow the fractionation and characterisation of all EV types released by cells. This is a critical first step towards defining the precise functionality of all secreted EV sub-populations. We report here a facile method for unbiasedly fractionating EVs released from the human colon cancer cell line LIM1863. Biophysical properties of two major EV subpopulations, including size, morphology, buoyant density, zeta potential, and protein composition are reported.