Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids.
Researchers from the University of Melbourne have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.
Schematic of the exosome isolation protocol from solid brain tissue
Fresh frozen (−80°C) human frontal cortex was sliced with a razor blade on ice while frozen to generate 1–2 cm long, 2–3 mm wide sections. The cut sections are dissociated while partially frozen in 75 U/ml of collagenase type 3 in Hibernate-E at 37°C for a total of 20 min. The tissue is returned to ice immediately after incubation and protease and phosphatase inhibitors are added. The tissue is spun at 300 × g for 5 min at 4°C (pellet is used as the brain homogenate + collagenase control), the supernatant transferred to a fresh tube, spun at 2000 × g for 10 min at 4°C, then at 10,000 × g for 30 min at 4°C. The EV-containing supernatant is overlaid on a triple sucrose cushion (0.6 M, 1.3 M, 2.5 M) and ultracentrifuged for 3 h at 180,000 × g to separate vesicles based on density. The top of the gradient is discarded and fractions designated 1, 2 and 3 are collected and the refractive index is measured. Each fraction is further ultracentrifuged at 100,000 × g to pellet the vesicles contained in each fraction. Each preparation is validated by a combination of techniques including electron microscopy and RNA and protein analysis. Note – some tissue samples will not be amenable to this method. Post-mortem delay, storage time and the number of freeze-thaw cycles will negatively impact on tissue quality and result in contamination of the fractions with cellular debris and non-exosome vesicles.