Harvesting EVs with conventional protocols, such as sequential centrifugation, has raised questions concerning the intactness and the purity of the EVs after purification. Herein, we propose a novel BE-SEC based purification of EVs which is fast, reliable and scalable.ĮV isolation with bind-elute size exclusion columns and characterization We show that the BE-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. Since EVs are larger than 700 kDa, they would be eluted directly in the flow through, while small soluble proteins and impurities less than 700 kDa would enter the core and remain trapped. We hypothesised that the BE-SEC column would be suitable for EV purification from cell culture conditioned medium (CM). The technology combines both size separation with bind-elute chromatography (BE-SEC) where large molecules bypass these beads while molecules smaller than 700 kDa penetrate the inert outer shell and bind to hydrophobic and positively charged octylamine ligands within the core.
In this article, we have evaluated a novel liquid chromatography technique for EV purification: using core bead chromatography. To overcome these issues, several other promising purification techniques have been proposed, such as precipitation kits 24, 25 and size exclusion chromatography (SEC) 19, 26. Furthermore, this technique is time consuming and prone to variable results due to the diverse protocols and equipment used in different laboratories 23. We and others have previously shown that the UC step damages the vesicles and leads to aggregation 18, 19, 20, which can ultimately affect downstream analysis 21 or application of EVs 19, 22. The gold standard in the field is to purify EVs by sequential centrifugation followed by an ultracentrifuge (UC) step to pellet the EVs at 110,000 × g 18. EVs are of fundamental importance in conveying critical intercellular messages 8, 10 both in physiological and pathological processes, such as taking part in the coagulation cascade 11, immune response 12, 13, 14 as well as aiding the spread of malignancies 9, 15 and viral infections 16, 17.īecause of their small size, physicochemical properties and the complexity of the surrounding fluid, purification of EVs is a great challenge. They carry proteins and RNAs, both miRNAs and mRNAs, and have been shown to transfer their cargo to recipient cells 3, 8, 9. 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. In this article, the term EVs will refer to exosomes and MVs only.
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.Įxtracellular 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. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. 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. 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.
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. 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.
0 kommentar(er)
