Analysis of Extracellular Vesicles Using Coffee Ring

Extracellular vesicles are categorized in subsets according to their biogenesis processes. To facilitate the investigation of subsets, an effective method is needed for isolating subpopulations. The efficacy of existing density and size-based isolation methods is limited, and as a result, the correlation of properties within separated subpopulations is modest. Here, we introduced size separation with ～48 nm resolution that exploits Marangoni flow and the coffee-ring effect in microdroplets in which extracellular vesicles are spatially deposited at different location according to size of extracellular vesicle. Interestingly, the analysis of tetraspanin proteins of the extracellular vesicles facilitated by this method reveals that the size of extracellular vesicles is correlated with expression of tetraspanin proteins (CD9, CD63, CD81) that are associated with the size of extracellular vesicles. The findings show that CD9 and CD81 are uniformly expressed regardless of size, CD63 is highly expressed only in larger extracellular vesicles. This evidence indicates that extracellular vesicles can be classified based on size and expression of CD63