Organs-on-a-chip are microfluidic devices containing cells in capillaries, through which a continuous flow of cell culture medium is maintained. By regulating concentration gradients, shear stresses and mimicking barriers and interactions between tissues, these systems enable in vitro simulation of physiological conditions of tissues and organs. Various types of these systems can be developed, including an endothelium-on-a-chip. Since the endothelium controls the permeability of the vascular wall and regulates vascular tone through the release of various substances, these systems enable studies of permeability and diffusion, effects of drugs on endothelial function, and pathophysiological processes associated with endothelial dysfunction. The aim of this study was to develop and optimize an endothelium-on-a-chip and to evaluate the impact of induced oxidative stress on human endothelial cells EA.hy926 under both static and flow conditions. Before integrating the chip into the microfluidic system, the functionality of all components of the system was examined, and the impact of different media on cell viability in atmospheric CO2 was evaluated. We determined the concentration of the cell suspension that ensured optimal cell confluency on the surface of the capillary and evaluated the effect of collagen coating on cell attachment rate. By increasing the medium flow through the capillary, we assessed the suitability of our system for simulating the physiological conditions of arterioles and arteries, and developed a method for determining cell viability within the capillary. After system optimization, oxidative stress was induced using hydrogen peroxide, and cell viability under static and flow conditions was compared. Our results demonstrated that the system operates in accordance with the Hagen Poiseuille law, and revealed the influence of hydrostatic pressure fluid flow within microfluidic system was observed. The on-chip experiments were performed with CO2-independent medium, which ensured the cell survival in a CO2-free atmosphere. The importance of the cell adaptation to the new medium and conditions without CO2 were also evaluated. For cell seeding in the capillary, we selected a cell suspension concentration that ensured immediate cell confluency with a minimal unattached. Coating the capillary surface with collagen accelerated the rate of cell attachment and improved cell adhesion to the surface. In induced oxidative stress, hydrogen peroxide exhibited higher toxicity under flow conditions compared to static ones. The endothelium-on-a-chip was successfully developed and optimized to the extent that it enables the cell viability measurements under various conditions.
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