Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes

Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.