Exosomes are small vesicles, ranging from 30-120 nanometers in diameter, secreted from cells throughout the human body. They are marked on their surface by proteins termed ‘tetraspanins’, aptly named as they contain four transmembrane proteins. Examples of these tetraspanins include CD9, CD45, CD63, and CD81, and they provide structural integrity to the exosome membrane. Exosomes facilitate in homeostasis by transferring active biologics from donor cells to recipient cells, modulating the recipient cells’ phenotype. Given their small size, exosomes can present daunting challenges in visualization. Modern techniques to view them include: nanoparticle tracking analysis, electron microscopy, and atomic force microscopy. We previously developed a novel pipeline for the production and isolation of exosomes to higher concentrations and purities than other currently employed methodologies. We wanted to expand upon this system by being able to fluorescently visualize intracellular maturation of exosomes from the donor cells and their subsequent uptake in recipient cells at the nanometer level. From this approach, we could have a fully tractable system from exosome biogenesis to endocytosis. To accomplish this, we created clonal cell lines expressing eGFP- or mCherry-conjugated CD9, CD63, CD81, and combinations thereof. These fluorescently tagged integral membrane proteins can be visualized and localized to high precision using super-resolution microscopy inside of the producer cell (i.e. during exosome biogenesis). Isolation of the exosome yields vividly bright pellets, and their addition to naïve cells can be visualized again using super-resolution microscopy. In summary, we have developed a nanometer-scale workflow for the visualization of de novo synthesized exosomes, their purification from cell-free fluids, and how their cargo is trafficked in recipient cells.

 

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