Abstract
Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.
Highlights
Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers
EVs were isolated from 200 μl of pooled human serum using differential centrifugation followed by either; ultracentrifugation (UC), polymer-based precipitation using Exoquick plus, size exclusion chromatography (SEC) using qEV columns or iodixanol density gradient centrifugation (DG) followed by ultracentrifugation
EVs are released into the bloodstream by cells under normal and pathological conditions and carry RNA, lipids, and proteins from their host cells that can represent the molecular composition of the parental cell
Summary
Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. EVs have been identified in a diverse range of human biofluids including serum, plasma, urine, saliva, breast milk, amniotic fluid, ascites fluid, cerebrospinal fluid and bile[7,8]. These EVs are classified into three groups; exosomes, microvesicles and apoptotic bodies depending on their size, biogenesis and method of cellular release. Serum and plasma are attractive sources of EV-based biomarkers as blood sample acquisition is a minimally invasive procedure and tumour cells release greater amounts of circulating EVs into the bloodstream[28,29]. Frozen biobanked serum is a valuable source of EVs for retrospective studies
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