Comparative Isolation and Multi-Omics Profiling of Brain Region-Derived Extracellular Vesicles Reveals Method-Dependent Variability
Extracellular vesicles (EVs), including exosomes and microvesicles, are lipid-bound particles that mediate intercellular communication within the central nervous system (CNS). Accurate isolation and characterization of brain-derived EVs are critical for understanding their roles in physiology and disease, as well as for biomarker discovery. Here, we compared four methods to isolate EVs from the cerebral cortex, hippocampus, and thalamus of mouse brains. Nanoparticle tracking analysis revealed that EV size distributions were consistent across regions and methods, while particle yield varied, with Method 1 producing the highest concentrations. Western blotting confirmed the presence of a canonical EV marker in particles isolated with Method 1 and 2, indicating successful vesicle enrichment. Although label-free proteomic profiling demonstrated a high degree of overlap between Methods 1 and 2 within the same brain region, Method 1 allowed to identify a higher number of unique proteins. These findings indicate that the isolation method impacts EV recovery and composition. Our study provides a framework for selecting and optimizing EV isolation protocols from brain tissue, facilitating reliable downstream analyses.  Furthermore, the study examines the applicability of selected method for downstream multi-omics analyses. The results demonstrate the effectiveness of the proposed protocol, highlighting the simplicity and efficiency of EV isolation with minimal laboratory requirements while confirming its compatibility with multi-omics approaches. Together, these findings underscore the value of our method for translational research and its potential to advance the study and application of EVs in diagnostic and therapeutic fields.
Extracellular vesicles (EVs), including exosomes and microvesicles, are lipid-bound particles that mediate intercellular communication within the central nervous system (CNS). Accurate isolation and characterization of brain-derived EVs are critical for understanding their roles in physiology and disease, as well as for biomarker discovery. Here, we compared four methods to isolate EVs from the cerebral cortex, hippocampus, and thalamus of mouse brains. Nanoparticle tracking analysis revealed that EV size distributions were consistent across regions and methods, while particle yield varied, with Method 1 producing the highest concentrations. Western blotting confirmed the presence of a canonical EV marker in particles isolated with Method 1 and 2, indicating successful vesicle enrichment. Although label-free proteomic profiling demonstrated a high degree of overlap between Methods 1 and 2 within the same brain region, Method 1 allowed to identify a higher number of unique proteins. These findings indicate that the isolation method impacts EV recovery and composition. Our study provides a framework for selecting and optimizing EV isolation protocols from brain tissue, facilitating reliable downstream analyses.  Furthermore, the study examines the applicability of selected method for downstream multi-omics analyses. The results demonstrate the effectiveness of the proposed protocol, highlighting the simplicity and efficiency of EV isolation with minimal laboratory requirements while confirming its compatibility with multi-omics approaches. Together, these findings underscore the value of our method for translational research and its potential to advance the study and application of EVs in diagnostic and therapeutic fields.
University of Modena and Reggio Emilia