Abstract

Everything We Think We Know About Extracellular Vesicle Cargo is Wrong

Terry Morgan, Professor, Oregon Health and Science University

Extracellular vesicles (EVs) comprise a range of submicron particles, including small EVs (exosomes ~75-150 nm) derived from endosomal biogenesis, larger microvesicles (< 1um) that bud directly from the cell membrane, and fragments of cells undergoing necrosis and/or apoptosis. These lipid encapsulated EVs contain surface cell-specific protein markers and a variety of RNAs that provide insights into their source and potential function (e.g. regulating angiogenesis and immune microenvironments).  Methods like density gradient ultracentrifugation followed by size-exclusion chromatography (SEC) provide a heterogeneous mixture of EVs from a variety of cell sources, and in a variety of sizes. Affinity capture lacks size-specificity, which is important because larger microvesicles are thought to have entirely different contents and biological functions than small EVs (exosomes). These methodological shortcomings have limited adequate EV profiling and characterization.  To address this need, the Morgan laboratory uses multiplex nanoscale fluorescent activated cytometric sorting (nanoFACS) with 40nm resolution to image, count, and isolate cell- and size-specific EVs. Importantly, this next generation approach provides highly efficient flow sorting of multiplex-labeled EVs at the 0.3 nanoliter droplet scale (5x better than commercially available FACS machines using our custom made sorting nozzle and EV-specific protocol).  We have completed validation studies characterizing flow sorted placental EVs according to guidelines put forth by the International Society of Extracellular Vesicles (ISEV), including nanoparticle tracking analysis, CryoEM, ELISA, and microRNA content within small EVs compared with flow sorted 100nm liposomes spiked into the same plasma samples. Spiked in liposomes are an important negative control for background contamination inherent in any EV isolation method and novel to our EV isolation approach.