Modern isolation and separation techniques for extracellular vesicles

The ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1–4, glycolytic enzymes and cytoskeletal proteins are absent from exosomes. We identify Annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular endosome-dependent, but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity. A reassessment of exosome composition establishes the differential distribution of protein, RNA, and DNA between small extracellular vesicles and non-vesicular extracellular matter and establishes that small extracellular vesicles are not vehicles of active DNA release.