Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma

Abstract

BackgroundPlasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.MethodsBy using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science).ResultsOur data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit.ConclusionsWe demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.Electronic supplementary materialThe online version of this article (10.1186/s12967-017-1374-6) contains supplementary material, which is available to authorized users.

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

“…In line with earlier publications, we demonstrate that precipitation- and sedimentation-based isolations co-fractionate significant amounts of serum albumin that mask genuine EV-enriched proteins. Additionally, we herein confirm previous findings [69,83,87] that SEC-based methods represent an efficient way of removing high-abundance serum proteins (Supplemental Figure 6), trading decreased vesicle yield for higher purity [88]. …”

“…These differences might be due to capturing different EV populations or manipulation of originally identical EVs during isolation by aggregation [67] or coating with serum proteins [68]. In line with our findings, Stranska et al recently reported larger particle diameters for EV samples isolated from human plasma by membrane affinity compared to SEC [69]. For most kits, variability of particle diameters was greater in sepsis samples, indicating disease-specific changes in circulating vesicles, or interferences caused by matrix effects in serum from critically ill patients.…”

“…Potentially owing to insufficient starting material or technical factors, and in contrast to preexisting publications [23,34,80], we did not detect TSG101 and CD81 for any isolation method. Similar findings were recently presented in a publication by Stranska et al, which demonstrated the absence of CD81 and TSG101 in plasma EVs isolated by membrane affinity [69]. Additionally, recent advances in the field have demonstrated that so-called exosome markers can also be present on other classes of EVs and that EV isolates are a heterogeneous mixture of various subpopulations with specific protein profiles [81,82].…”

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

“…In many instances maintaining the physical integrity of EVs and their surfaces during preparation represents an important pre‐analytical consideration, especially with low abundance material. In this regard, the standard ultracentrifugation methods are often being complemented or replaced by gentler approaches such as density gradient centrifugation, sucrose cushion centrifugation, gel‐permeation chromatography, size‐exclusion chromatography, affinity capture, or microfluidics …”

Effective enrichment of urinary exosomes by polyethylene glycol for RNA detection

“…They are suitable for most laboratories and easy to be utility, however, they are costly, especially if a large number of biological samples need to be processed. Besides that, various techniques including filtration [20][21][22], microfluidic-based method [23,24], immuno-affinity and peptide-based isolation [25], size-exclusion chromatography [26][27][28], and so on, have also been proposed for enrichment of exosomes. They have advantages and disadvantages, can be selected according to the experimental command.…”