Harvest Time-Dependent Changes in the Composition and Function of Microglia-Derived Small Extracellular Vesicles.

Microglia are dynamic macrophage-like cells that survey the central nervous system (CNS) and mediate neuroinflammatory responses. Even under homeostatic conditions, they exhibit phenotypic plasticity in response to environmental cues. Microglia-derived small extracellular vesicles (sEVs), which are lipid bilayer vesicles encapsulating proteins, lipids, and nucleic acids, reflect the physiological state of their parent cells and influence recipient cell function. Although increasing attention has been given to their roles in neuroinflammation, neurodegeneration, and intercellular communication, the impact of experimental variables, particularly harvest timing, on microglial sEV composition remains poorly understood. This study investigated how different harvest durations (24, 48, and 72 h) influence the molecular and functional properties of sEVs released by BV2 microglial cells. sEVs were isolated via ultracentrifugation and characterized for particle size, yield, RNA and protein content, surface marker expression, and proteomic profile. While sEV size and morphology remained consistent across time points, particle yield and RNA content increased significantly with prolonged harvest, peaking at 72 h. Classical sEV markers (CD9, CD81, TSG101) were consistently expressed, suggesting stable vesicle identity. However, proteomic profiling revealed a time-dependent shift in cargo, with an increase in unique proteins and enrichment of stress- and neurodegeneration-related pathways at 72 h. Functional assays further demonstrated that sEVs collected at this time point reduced viability in both microglial and neuronal cells. Together, these findings highlight harvest time as a critical determinant of microglial sEV composition and bioactivity, with implications for their use in diagnostics, therapeutics, and drug delivery applications.