The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens

Abstract

BackgroundRobust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community ‘gold standard’ for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification.Methodology/Principal findingsTransmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs.Conclusions/SignificanceThe data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells

“…Similar to the published finding of Xu's team, our data suggested that human SAE cells are able to secrete EVs under control and cigarette smoke exposure conditions in an in vitro model. Although the differential centrifugation technique is widely used for the EV purification, we opted for the two-step procedure, i.e., precipitation reagent solution and size exclusion chromatography (SEC), as it is associated with a higher purity of EVs than differential centrifugation alone [43]. We found that CSC exposure did not cause significant differences in the concentration and size of SAE-derived EVs.…”

Extracellular Vesicles in the Tumour Microenvironment: Eclectic Supervisors

“…TDE have been proposed as biomarkers, diagnostics and therapeutics, or "theranostics" [152] (Table 2). Although the EV isolation procedure is still a matter of debate, currently, the gold standard for EV purification includes differential centrifugation [153,154]. Differentially expressed proteins and genetic materials in EV recovered from tumour patients and healthy subjects are the so far most applied approach for biomarker discovery.…”

Extracellular Vesicles as an Efficient and Versatile System for Drug Delivery

“…As EVs are heterogeneous, different isolation techniques and variations in equipment settings would enrich certain EVs subtypes while depleting others [ 158 ]. The level of EVs purity also varies, thus giving rise to difficulties in experiment reproducibility and quality control [ 159 , 160 , 161 ].…”