Laminar Flow Alters EV Composition in HUVECs: A Study of Culture Medium Optimization and Molecular Profiling of Vesicle Cargo.

Endothelial cells (ECs) experience shear stress associated with blood flow. Such shear stress regulates endothelial function by altering cell physiology. Since most cell culture protocols and media compositions are designed for static cultures and experiments with ECs are predominantly conducted under these non-physiological conditions, a model for culturing ECs under flow conditions is developed, which more closely mimics their physiological environment. This approach also enables the isolation of EVs while minimizing FCS-derived contaminants. In this study, a comprehensive assessment of how physiologically relevant cultivation conditions influence the vesicle composition and function of ECs is provided. A detailed investigation is conducted for the effect of different cell culture media on morphology and marker expression of human umbilical cord endothelial cells (HUVECs) and EVs, and optimize the conditions to culture ECs under flow, tailoring them specifically to facilitate the efficient isolation of EVs using a hollow-fiber system model. These EVs are then characterized and compared to those isolated from traditional static culture conditions. Overall, this study presents a model on isolating EC-derived EVs under conditions that closely mimic physiological environments, and characterization at their proteome, gene expression, and microRNA profile.