Improving extracellular vesicle isolation from non-mammalian blood plasma : enhancing efficiency through adjusted osmolarity

Extracellular vesicles (EVs) are membrane-bound particles released by cells. They play an essential role in intercellular communication by transporting RNA, DNA, proteins, and lipids. While the biology of mammalian EVs is well characterized, comparatively little is known about EVs in non-mammalian vertebrates. This gap limits our understanding of the evolutionary conservation and functional diversity of EV-mediated signaling. In this study, we present a standardized and broadly applicable protocol for isolating EVs from the blood plasma of four representative non-mammalian vertebrate species: chicken (Gallus gallus domesticus), corn snake (Pantherophis guttatus), African clawed frog (Xenopus laevis), and common carp (Cyprinus carpio L.). The protocol, based on ultracentrifugation, offers a practical and accessible method without the need for specialized equipment or commercial kits. By facilitating the study of EVs across diverse vertebrate taxa, this work contributes to the broader understanding of EV biology, including their potential roles in evolutionarily conserved signaling pathways and virus transmission.