Isolation and Characterization of Small Extracellular Vesicles Derived from Epicardial Adipose Tissue.

Epicardial adipose tissue (EAT) is a distinct visceral adipose depot located between the myocardium and the visceral layer of the pericardium. Owing to the absence of an anatomical barrier between EAT and the heart, EAT can communicate with the myocardium through paracrine and vasocrine mechanisms. Small extracellular vesicles (sEVs) secreted by EAT, which are lipid bilayer-enclosed nanoparticles that mediate intercellular communication by transferring bioactive cargoes, including proteins, lipids, and nucleic acids, to recipient cells, may serve as key mediators of this crosstalk. However, the extraction of sEVs from EAT remains technically challenging. In this study, we established a reproducible protocol for isolating sEVs from rat EAT using collagenase digestion followed by differential ultracentrifugation. The isolated vesicles were characterized by transmission electron microscopy, nano-flow cytometer, and western blotting. This protocol provides a practical and reproducible experimental framework for isolating EAT-derived sEVs and may facilitate future studies exploring their roles in cardiovascular physiology, disease mechanisms, biomarker discovery, and therapeutic development.