Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

Abstract

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

“…Relative success of these different methods in terms ofrecovery and specificity to EVs (as compared to non-vesicular components), or to EV subtypes, has been addressed in a previous ISEV Position Paper (see Figure 1 of [56]), and is summarized in Table 1 below. To achieve better specificity of EV or EV subtype separation, most researchers use one or more additional techniques following the primary step, such as washing in EV-free buffer, ultrafiltration, application of density gradients (velocity or flotation), or chromatography [6,[99][100][101][102].…”

Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source

“…(2) intermediate recovery and intermediate specificity: methods include size-exclusion chromatography (Stranska et al, 2018) and high molecular weight centrifugal filters (Vergauwen et al, 2017); (3) low recovery and high specificity: methods include filtration combined with SEC (Th ery et al., 2018), immunoaffinity capture-based techniques, and microfluidics-based isolation techniques; (4) high recovery and high specificity:…”

The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos

“…Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) was washed three times with PBS containing 0.32% trisodium citrate dihydrate (ChemCruz, Dallas, TX, USA) [22,23]. For the preparation of one column, nylon net with 20 µm pore size (NY2002500, Merck Millipore, Billerica, MA, USA) was placed on the bottom of a 10-mL syringe (BD Biosciences, San Jose, CA, USA), followed by stacking of 10 mL Sepharose CL-2B.…”