Human Endothelium-on-a-Chip: Development of a Microfluidic Model for Cell Viability Assessment Under Oxidative Injury

Endothelial responses to oxidative stress are influenced by the presence of blood flow and shear stress. To better capture these conditions in vitro, we developed an endothelium-on-a-chip model for the study of acute oxidative injury in EA.hy926 cells under flow. We adapted the culture conditions for use outside a conventional 5% CO₂ atmosphere, and we improved endothelial adhesion and retention under flow by collagen coating of the chip surface. Our model tolerated shear stress of up to 0.89 Pa, which corresponds to the in vivo values experienced by endothelial cells in the aorta and venous system. On this model, we designed an acute oxidative injury protocol based on high concentrations of hydrogen peroxide (H₂O₂) as an endogenous source of oxidative stress and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as an exogenous molecule. The inclusion of flow increased the apparent sensitivity of the model to oxidative injury compared to static conditions, as the IC₅₀ values decreased from 16.2 mM to 12.0 mM for H₂O₂ and from 100.7 mM to 72.5 mM for AAPH under flow conditions. Pretreatment with quercetin (1.5 µM) reduced AAPH-induced oxidative injury, thus showing the potential applicability of the model for antioxidant screening assays.