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

Chelsea N Davis,Martin T Swain,Toby J Wilkinson,John J Tomes,Russell M Morphew,Helen Phillips,Peter M Brophy,Gabriel Rinaldi

doi:10.1371/journal.pntd.0007191

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.

Highlights

Extracellular vesicle (EV) purification is challenging to standardise due to the diversity of sample composition producing extracellular vesicles (EVs), the need for high recovery of functional EVs, the quality of EV preparation and the simplicity of isolation [1,2,3,4,5]
It is important that the optimal EV purification method is realised in order to obtain high quality EVs to have confidence in understanding EV biology and function
The zoonotic parasite, Fasciola hepatica, was cultured as a case study to investigate the importance of EV purification from helminth culture media

Summary

Introduction

Extracellular vesicle (EV) purification is challenging to standardise due to the diversity of sample composition producing EVs (cell cultures and body fluids), the need for high recovery of functional EVs, the quality of EV preparation and the simplicity of isolation [1,2,3,4,5]. The most widely accepted method to isolate EVs involves differential centrifugation (DC). This method encompasses sequential centrifugations, increasing in speed and time, to pellet particles decreasing in size [1,2,4,5,6,7,8,9,10,11]. To overcome EV purification challenges, many downstream EV investigations require further isolation procedures to accommodate DC, to improve sample purity and validate research analysis. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). 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

Methods

Results

Discussion

Conclusion