Sequential Filtration: A Gentle Method for the Isolation of Functional Extracellular Vesicles

Abstract

A prevalent challenge in isolating extracellular vesicles (EVs) from biological fluids is the reliable depletion of abundant contaminants—including free proteins and biomolecules, as well as nontarget vesicle subpopulations and other nanoparticulates—from the sample matrix while maximizing recovery. Sequential Filtration is a recently published approach for the size-based isolation of exosomes that is ideally suited for large-volume biofluid samples such as ascites , urine , lavage fluid, or cell-conditioned media. We describe a straightforward, three-step protocol comprising back-to-back steps of dead-end (normal) filtration, tangential-flow filtration, and track-etched membrane filtration that can be applied to yield a homogeneous population of exosome-sized extracellular vesicles. The approach is scalable and employs relatively gentle manipulation forces to fractionate and concentrate extracellular vesicles with good purity and functional integrity.