Isolation and characterization of the morphology, size and particle number of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) cell line derived large and small extracellular vesicles.

Extracellular vesicles (EVs) are 50-1,000 nm lipid bilayer-bound vesicles, released into the extracellular environment by various cell types for intercellular communication purposes. The quantitative and qualitative characteristics of EVs can be affected by stress and pathological conditions. The majority of extracellular vesicle (EV) studies have been performed on mammalian cell lines or bodily fluids. EVs have been previously described from bodily fluids like plasma, serum or mucus in different fish species, however the available knowledge of fish cell line derived EVs is limited and in the vast majority of studies, the overall focus is on small EVs (< 200 nm). We isolated large and small extracellular vesicles from zebrafish (Danio rerio) liver (ZFL), rainbow trout (Oncorhynchus mykiss) liver (RTL-W1), gill (RTgill-W1) and intestinal epithelial (RTgutGC) cell lines using stepwise centrifugation and characterized the size and morphology of EVs. Here we demonstrated that large and small extracellular vesicles can be successfully isolated using stepwise centrifugation from the serum-free medium of the selected piscine cell lines after a 24-h incubation period. The size distribution of large and small EVs isolated from the piscine cell lines suggest that large and small EV groups show high diversity in size ranges, containing heterogenous subpopulations in sizes, and the results highly depend on the applied method and whether filtration steps were included following the isolation. The spherical morphology of EVs was verified by transmission electron microscopy.