Cell suspension cultures (CSCs) from Coffea arabica L. have recently been established in our laboratory (Di Bonaventura et al. 2024). In addition to being a valuable source of secondary metabolites, these CSCs may also release extracellular vesicles (EVs) into the culture medium. Following filtration of the CSCs, EVs were isolated by differential centrifugation, first at 100,000 × g for 36 minutes (fraction 100k g), and then at 125,000 × g for 6 hours (fraction 125k g). The two EV fractions were very similar in terms of size, shape, and ζ-potential. SDS-PAGE and Western blot analyses revealed the presence of H+-ATPase and calnexin 1/2 in both fractions, serving as markers for the plasma membrane and endoplasmic reticulum, respectively. Proteomic analysis identified nearly 500 proteins in the 100k g fraction and approximately 300 in the 125k g fraction. Of these, 232 proteins were shared by both fractions, while 267 and 71 proteins were unique to the 100k g and 125k g fractions, respectively. STRING analysis indicated that the 232 shared proteins were predominantly enriched in the extracellular region. The 100k g fraction was enriched in proteins associated with the cell periphery and plasma membrane, whereas the 125k g fraction was primarily enriched in proteins localized to the extracellular region. These findings demonstrate that CSCs from C. arabica represent a valuable and scalable platform for the production of EVs, which may have potential biotechnological applications.
