Influence of species and processing parameters on recovery and content of brain tissue‐derived extracellular vesicles
Year: 2020
Type: article
Abstract: Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,410
Funders: Michael J. Fox Foundation for Parkinson's Research, Johns Hopkins University, La Trobe University, National Institutes of Health, Medical Research Council, National Health and Medical Research Council, National Institute on Aging, National Institute of Mental Health, National Institute on Drug Abuse, NIH Office of the Director, National Cancer Institute, National Institute of Allergy and Infectious Diseases, National Center for Research Resources, Office of Strategic Coordination, Office of Research Infrastructure Programs, National Institutes of Health, Common Fund
Awards: G2305452004, G2344771908, Extracellular Vesicle-Mediated Immunomodulation in HIV Infection and Nicotine Abuse, G3879737662, G4215931020, Novel Approaches to Capture, Sorting, and Characterization of CNS-Origin Extracellular Vesicles, Biomarkers to aid clinical trials for neurodegenerative disease, G5577482043, G6079011624, G6585641498, G7699446225, Discovering novel players in mechanisms of extracellular vesicle release, cargo loading, and early pathogenesis of late-onset Alzheimer's Disease, G8144267149, Development of an SPF Macaca nemestrina Breeding Colony, Novel Separation Methods for exRNA Carriers: Extracellular Vesicles, Lipoprotein Particles, and Protein Aggregates, Next-generation extracellular vesicle biologics to target central nervous system and peripheral reservoirs of HIV, G8773427133, G9000156707, Extracellular Vesicle and Extracellular RNA Biomarkers of HIV-1 Central Nervous System Pathogenesis and Cigarette Use, Continued support for an SPF Macaca nemestrina breeding colony to provide animals to NIH funded HIV research projects, Johns Hopkins Alzheimer's Disease Research Center, Novel Approaches to Capture, Sorting, and Characterization of CNS-Origin Extracellular Vesicles
OpenAlex ID: W3039855774