Recent advances in modern extracellular vesicle isolation and separation techniques

Since slightly sluggish beginning some twenty years ago, heterogeneous nanosized extracellular vesicles (EVs), secreted by almost all mammalian cells, all Gram-negative and Gram-positive bacteria and several plant tissues, have grabbed an enormous attention especially during the last years due to their functional role in intercellular and interorgan communications, offering possibilities for great innovations in diagnostics and therapeutic monitoring and prognosis. The availability of their reliable isolation and separation techniques that can conquer most of the challenges related to purity, yield, scalability, integrity, nanoscale contaminants, operation time, and complex sample matrices is a crucial prerequisite for the exploitation of EVs and their subpopulations. The utmost goal of this review, that is a continuation of our overview published in 2021, is to introduce the most recent modern and emerging isolation and separation approaches and trends, with their specific properties and applications. The focus is again on size-, charge-, and affinity-based techniques and ultracentrifugation and precipitation-based techniques are included only for comparison purposes. The isolation techniques will be briefly compared with each other in terms of different important parameters. Furthermore, a few industrial promising applications are introduced. Although in the newly developed isolation and separation techniques and methods especially the presence of interfering nanocomponents, causing lower purity, has been much better minimized or even eliminated, and although even in vivo studies have more frequently been carried out, it is obvious that only combined and hyphenated techniques can overcome at least most of the challenges.