Abstract
Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.
Highlights
Extracellular vesicles (EVs) are nanosized cell-derived vesicles[1,2,3] delimited by a lipid bilayer and typically divided into three subgroups, according to their biogenesis pathways; exosomes, microvesicles (MVs) and apoptotic bodies[4]
We show that the bind-elute size exclusion chromatography (BE-size exclusion chromatography (SEC)) method is suitable for purification of EVs, with yields consistently reaching 80% and vesicular purity comparable to the gold standard method in the field
The beads used in the BE-SEC column are designed to trap molecules smaller than 700 kDa and allow larger particles to pass through
Summary
Extracellular vesicles (EVs) are nanosized cell-derived vesicles[1,2,3] delimited by a lipid bilayer and typically divided into three subgroups, according to their biogenesis pathways; exosomes, microvesicles (MVs) and apoptotic bodies[4]. Exosomes are 70–150 nm in size and originate from the endocytic pathway[5] whereas MVs are generally larger, 100–1000 nm in diameter and bud directly from the plasma membrane[6, 7] They carry proteins and RNAs, both miRNAs and mRNAs, and have been shown to transfer their cargo to recipient cells[3, 8, 9]. We and others have previously shown that the UC step damages the vesicles and leads to aggregation[18,19,20], which can affect downstream analysis[21] or application of EVs19, 22 This technique is time consuming and prone to variable results due to the diverse protocols and equipment used in different laboratories[23]. We propose a novel BE-SEC based purification of EVs which is fast, reliable and scalable
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