A method for isolating small extracellular vesicles from parenchymal tissues

Abstract

Extracellular vesicles (EV) are released by almost all cells into the extracellular space, where they navigate through the interstitium or transfer bioactive molecules into neighboring cells, thereby regulating tissue homeostasis. Small EV (sEV) generally refer to EV with a diameter of less than 200 nm, which include exosomes formed in the endosomal system and small ectosomes assembled at and released from the plasma membrane. While techniques for isolating sEV from large body fluids auch as blood and urine are relatively well-developed, isolating sEV from solid organs remains challenging. This is because the preparations are often seen contaminated with intracellular vesicles due to cell breakage resulting from the treatments used to release sEV from the extracellular matrix. Here, we introduce a new gentle method that allows for the reliable isolation of sEV from heart and liver tissues with high purity and yield. The protocol involves an initial treatment of tissues with selected collagenase, followed by consecutive differential centrifugation and density-gradient ultracentrifugation to separate sEV from cells and large EV (lEV), and further density-gradient ultracentrifugation to fractionate sEV subpopulations. Characterization of the preparations reveals markedly enhanced cellular survival and reduced co-purification of extracellular non-vesicular particles as well as subcellular organelles. Moreover, we found that sEV isolated from heart, liver, and plasma are similar in morphology and size, but differ in density and protein marker distributions among sEV subpopulations. Our method may facilitate the isolation of sEV from solid tissues with better quality for further molecular characterization and functional studies.