Extracellular vesicle analysis

Cells release small, phospholipid membrane-enclosed particles, collectively referred to as extracellular vesicles (EVs), into their surroundings to enable intercellular communication. EVs have numerous functions in physiological and pathophysiological processes and show considerable promise for diagnostic and therapeutic applications. Technologies have rapidly evolved over the past two decades, providing a powerful, versatile toolset for preparing and characterizing EVs to facilitate research and translational efforts. However, considering the plethora of methods available, it is challenging to understand what makes one method more suited for a given experiment than another. The heterogeneity of EVs as well as the diversity in composition of their surroundings further add to this challenge. This Primer provides guidance for EV analysis across ecosystems, including accessible body- and environment-derived sources. We summarize the multi-step process of EV preparation, cover the guiding principles and considerations when performing and interpreting EV experiments, and reflect on the limitations and challenges in the fields of fundamental biology, biomarker development and therapeutic strategies.