Sub‐Neuronal Network Profiling of Extracellular Vesicle Release Using a Compartmentalized Neurofluidic Platform

ABSTRACT Extracellular vesicles (EVs) are membrane‐bound vesicles that are secreted by a wide range of organisms and cells, carrying cell‐specific receptors and molecular cargo such as proteins and nucleic acids. EVs have emerged as promising biomarkers for cancer and neurodegenerative disorders like Alzheimer's Disease (AD). Traditional methods for isolating neuron‐derived EVs from bodily fluids or conditioned media are based on bulk analysis methods, such as ultracentrifugation, isolation reagents, and immunoaffinity‐based techniques, and lack spatial resolution to capture localized secretion dynamics. Here, our neurofluidic platform compartmentalizes neuronal networks and enables spatially resolved analysis of EV profiling before subsequent traditional isolation and content screening. This intermediate resolution provides critical insights into localized sub‐neuronal EV secretion dynamics in cortical, hippocampal, and brainstem neurons. Using our platform, the influence of growth environment, cell maturation time, and exogenous stressors such as shear and biochemical stress can be unraveled. Biochemical stress is induced through okadaic acid (OA), a PP1A/PP2A inhibitor, which leads to hyperphosphorylation of proteins. In parallel, microRNA expression profiles are shown after OA treatment in primary neuron cultures, indicating an additional transcriptional response. These findings reveal regional differences in EV secretion dynamics associated with neuronal development and external stressors, including shear forces and PP1A/PP2A inhibition.