Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct

•Sequential centrifugal ultrafiltration affords unbiased isolation of extracellular vesicles.•Two major EV subtypes are secreted by colon cancer tumour cells – sMVs and exosomes.•sMVs (30–1300nm) are heterogeneous, whereas exosomes are homogeneous (30–100nm) in size.•Exosomes, unlike sMVs, contain protein markers Alix/TSG101/CD63/CD81.•350 proteins uniquely enriched in sMVs (not seen in exosomes) are potential sMV markers. Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ∼7.2×109 LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC–MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes; one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30–1300nm, the other (fraction Fn5) being homogeneous EVs of 30–100nm diameter; based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix+, TSG101+, CD81+, CD63+), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000g, DC-sMVs) and exosomes (100,000g EVs depleted of 10,000g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18–1.19g/mL and exosomes 1.10–1.11g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the