Considerations and Implications in the Purification of Extracellular Vesicles – A Cautionary Tale

Abstract

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into all biological fluids. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. As a result, there is growing interest in studying EVs in pathological conditions, including central nervous system (CNS)-related diseases, as EVs may be used for diagnostic purposes or as therapeutic agents. However, one major bottleneck is the need for better EV purification strategies when considering complex biological sources such as serum/protein-rich media or plasma. In this study, we have performed a systematic comparison study between the current gold-standard method: ultracentrifugation, to an alternative: size-exclusion chromatography (LC), using induced pluripotent stem cell (iPSC) derived complex media as a model system. We demonstrate that LC allows for derivation of purer EVs from iPSCs, which was previously impossible with the original UC method. Importantly, our study further highlights the various drawbacks when using the conventional UC approach that lead to misinterpretation of EV data. Lastly, we describe novel data on our iPSC-EVs; how they could relate to stem cell biology and discuss their potential use as EV therapeutics for CNS diseases.

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

“…Similar results have been observed recently by O’Neil [ 18 ] using ultrafiltration followed by SEC to isolate EVs from ovine uterus. In the same line, isolation by SEC has resulted in higher EVs recovery and significantly less protein contamination than EVs isolated by precipitation [ 36 , 81 ].…”

Impact of Culture Medium on Cellular Interactions in in vitro Co-culture Systems

“…Subsequently, EVs might be used to study cellular interactions in vitro omitting the requirement for a co-culture experiment and thus overcoming co-culture challenges. The biggest challenge of using EVs lies within the development of purification and characterization protocols (Lee et al, 2019).…”

(Sub)populations of extracellular vesicles released by TNF‐α –triggered human endothelial cells promote vascular inflammation and monocyte migration

“…In addition, specific tools for the isolation of disease‐associated EV (sub)populations are missing in the field [12]. Most likely, there are additional EV subpopulations present in both size‐ or density‐based separated EV fractions that are functionally more related to a certain disease [13]. The purity, and hence the functionality of EV subpopulations are still under debate [12,14].…”