Long‐Term Stability of Bacterial Extracellular Vesicles Stored at Different Temperatures

ABSTRACT Extracellular vesicle (EV) research has revealed EV involvement in a variety of biological processes, with potential applications in both fundamental research and therapeutic development. EVs of bacterial origin have been implicated in host‐immune interactions in the development of a variety of diseases. Despite differences in the membranes of bacterial and mammalian cells, past studies on EV storage conditions have focused on mammalian EVs. In this work, we evaluated the effects of storage temperature on the properties of EVs derived from gram‐positive ( Bifidobacterium longum ) and gram‐negative ( Escherichia coli ) bacteria and stored for a period of 8 weeks. It was hypothesized that storage over time would impact the integrity of the EV samples, particularly at higher temperatures. B. longum EV identity was confirmed by western blotting for lipoteichoic acid, GroEL, and RecA, while E. coli EV identity was confirmed by western blotting for lipopolysaccharide, GroEL, and Flagellin. Using nanoparticle tracking analysis (NTA), we observed no effect of storage on E. coli EV size or concentration but found that storage at 4°C for 4–8 weeks led to increases in B. longum EV size. No significant changes in particle size or concentration after storage were observed with microfluidic resistive pulse sensing (MRPS). An apparent increase in macrophage activation by the EVs was observed after 4 weeks of storage, followed by an apparent decrease in macrophage activation after 8 weeks of storage. Although EV protein concentration was stable with storage, the intensity and definition of some protein bands in gel electrophoresis appeared to decrease over the storage period. Overall, even short‐term storage of bacterial EVs led to changes in EV functionality, and size measurements alone were not necessarily indicative of EV stability.