Comparison of separation methods for immunomodulatory extracellular vesicles from helminths

Abstract

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminthderived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.

Compositional profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging

“…As expected, most EVs in both samples tend to cluster around a characteristic CA value with no dependence on their Deq (Figure 4a), suggesting that these nanoparticles carry the same nanomechanical fingerprint, i.e., similar stiffness independently of their size. Notably, the same result was also previously obtained on different EV populations deposited on substrates identical to those used in this study [Borup 2022]. Due to this, it is possible to define a zone on the CA/Deq plot which is typical of most pressurized EVs.…”

“…Recently, atomic force microscopy (AFM)-based nanomechanics have been established as a valuable tool to assess EV identity, purity and function [LeClaire 2021]: EVs have been shown to display a specific nanomechanical fingerprint which is detectable via force spectroscopy [Vorselen 2017; Piontek 2021] and which can be leveraged to discriminate between different EV populations [Sorkin 2018; Vorselen 2018]. We recently implemented a single-particle nanomechanical screening based on quantitative AFM morphometry which considerably increases analytical throughput [Ridolfi 2020a], making it possible to rapidly detect co-isolated, non-vesicular contaminants [Ridolfi 2020a; Ridolfi 2020b] and to give an estimate of their abundance relative to that of EVs [Borup 2022]. Conc4versely, the nanomechanical behaviour of LPs is still largely uncharacterized, the first studies having appeared only very recently [Baraimukov 2022].…”

Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species

“…Furthermore, the density of the fractions was measured by ELISA. In addition, the particle concentration (particles/ml) and sizes (nm) were measured by tunable resistive pulse sensing on qNano (Izon Science) and by nanoparticle tracking analysis on ZetaView (ParticleMetrix) ( Borup et al., 2022 ). For further approaches, Optiprep ® density gradient (ODG) fractions were pooled with a density in an optimal range for helminth EVs (1.09-1.12 g/mL), which corresponds also to the highest protein concentrations.…”

Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility

“…Thus, it is encouraged that further measures should be routinely implemented to ensure E/S preparations are not contaminated by bacterial or host contaminants. For instance, common bacterial contaminants can be detected by performing Mycoplasma testing, endotoxin testing or assaying for bacterial growth (Borup et al, 2022;Ridolfi et al, 2020). Furthermore, any procedures designed to reduce contamination should also be noted (e.g., if media from the first several hours of culture are excluded from analyses or if (and how) host cells have been removed).…”