Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues

Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space.

Researchers from the Nathan S. Kline Institute for Psychiatric Research describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin.

Overview of the procedure used to isolate brain EV subpopulations

a, Schematic representation of the procedure used to isolate EVs from the right murine hemibrain. b, Representative photographs of the most important steps during brain EV isolation (Steps 15–40). c, Representative photographs of the most important steps during the separation of brain EV subpopulations (Step 41A–B). A sucrose low-resolution gradient (left) and an iodixanol high-resolution gradient (center) loaded with brain EVs are shown before and after the overnight ultracentrifugation at 200,000g. The right panel shows the collection of the iodixanol EV fractions and the appearance of the EV pellets before resuspension. All animal procedures were performed following the National Institutes of Health guidelines with approval from the Institutional Animal Care and Use Committee at the Nathan S. Kline Institute for Psychiatric Research.

The researchers also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, they compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.