Isolation and Characterization of Small Extracellular Vesicles from Infrapatellar Fat Pads of Osteoarthritis Patients.

Extracellular vesicles (EVs), particularly small extracellular vesicles (sEVs) defined as vesicles smaller than 200 nm, serve as essential mediators of intercellular communication and released by nearly all cell types into various biological fluids. The infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA). However, standardized methods for isolating sEVs directly from IPFP explants remain limited. We present a reproducible protocol for the isolation and characterization of sEVs derived from IPFP tissues obtained from OA patients. Conditioned medium collected from IPFP tissue cultures is sequentially cleared of cells and debris by low-speed centrifugation, followed by ultracentrifugation to enrich vesicles within the sEV size range. The resulting vesicles are characterized according to minimal information for studies of extracellular vesicles (MISEV2023) recommendations using a set of representative protein markers across distinct functional categories: CD63 as a membrane-associated tetraspanin, ALIX and TSG101 as cytosolic proteins involved in biogenesis mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, and Calnexin as an endoplasmic reticulum-resident protein to monitor potential non-vesicular contamination. This workflow yields well-defined sEV preparations suitable for downstream applications such as functional assays or proteomic profiling in osteoarthritis research.