Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

Abstract

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

“…To test this hypothesis, we isolated EVs from 25 µL of plasma from two lung adenocarcinoma subjects and one healthy control using a Total Exosome Isolation kit. Note, that isolation of EVs using the precipitation reagent presented in the kit does not separate exosomes from microvesicles [17]. Next, the amount of DNA was analyzed by qPCR using mtCOXIII and nuACTB primers.…”

Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro

“…SEC-output fractions of 500 µL each were collected. The fractions with highest abundance of EVs but with the least protein contaminants are fractions 7 to 10 according to the product manual and our previous findings [ 27 ]. For each replicate, the EV fractions (i.e., 7 to 10) of five SEC column isolations were combined and concentrated from 10 mL to 200 µL.…”

“…Because of the known origin of the cell line, the disease specificity of CCM EVs is high, providing an opportunity to identify cancer-specific EV biomarker candidates. Compared with patient samples such as plasma [ 27 ], serum-free CCM contains relatively low contamination following EV isolation. The role of EVs in RCC has been studied in CCM [ 12 – 14 , 21 , 24 ], but no benign kidney cells were included to enable specific biomarker discovery.…”

Urinary Extracellular Vesicles: Uncovering the Basis of the Pathological Processes in Kidney-Related Diseases

“…This different approach separates EVs according to their flotation densities, together with their different sizes [ 53 ]. Top-to-bottom or bottom-to-top loading procedures can be applied to separate particles, along with the different densities of the solution [ 54 ], including different types of urinary EVs [ 55 ]. Like differential ultracentrifugation, this procedure is time-consuming.…”