Efficient Isolation and Characterization Methods for Chicken Serum Extracellular Vesicles

Extracellular vesicles (EVs) are small cell membrane-derived vesicles that are known as mediators of cell-to-cell communication. Efficient methods for chicken serum EV isolation and identification have not been established to satisfy the international guidelines of the Minimal Information for Studies of Extracellular Vesicles (MISEV). Therefore, the present study isolated EVs from chicken serum using four conventional methods-ultracentrifugation (UC), two polymer-based precipitations (PP), and filtration (FL)-and characterized them by western blotting (WB) using polyclonal antibodies (anti-CD9, anti-PDCD6IP, anti-TSG101, and anti-calnexin), nanoparticle tracking analyses, and transmission electron microscopy of MISEV guidelines. As a result, all isolated samples represented specific EV marker proteins, small particles sized 30-150 nm, and spherical morphology with a double membrane structure. In particular, FL-isolated EVs showed the most distinct expression of the marker proteins by WB analysis and the highest expression of miR-451, which is a stable microRNA of EVs, by quantitative PCR, suggesting that FL could isolate highly purified EVs. In conclusion, we established an optimal characterization method for chicken EVs and estimated the efficiency of EV isolation methods from chicken serum.