Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles

SIMPLE SUMMARY: Cetaceans are species of scientific interest for many reasons. First, they can be useful to assess environmental health and, second, they have peculiar features which also make them interesting for human comparative pathology. In the last decades, extracellular vesicles have been studied as important carriers in cell-to-cell communication, and many studies in human and veterinary medicine have focused on their role in pathophysiological mechanisms or as biomarker to diagnose diseases. In vitro studies are good models to explore extracellular vesicles. However, cell lines have been poorly used and investigated in these species. For these reasons, here we describe for the first time the isolation of extracellular vesicles from two cetacean cell lines established from bottlenose dolphin and Cuvier’s beaked whale. We also compare two different techniques to isolate extracellular vesicles, reporting the difference in the yield and quality of the obtained sample. This preliminary study on extracellular vesicles isolated in vitro aims to be the basis for future research to deepen our understanding on cetacean pathophysiology. ABSTRACT: Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier’s beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-β. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier’s beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.