Development of a 3D in vitro model to investigate sprouting angiogenesis under hydrostatic pressure stimulation

Angiogenesis is tightly regulated by the mechanical and biochemical cues of the microenvironment. To investigate the mechanobiological regulation of endothelial cells, we developed a 3D in vitro bioreactor that enables precise, leak-free application of hydrostatic pressure to endothelialized collagen I channels. Using this platform, we seeded primary human umbilical vein endothelial cells and primary human aortic endothelial cells at optimized densities and achieved reproducible channel endothelialization. We demonstrate that sufficient protein and RNA can be extracted from single channels, enabling downstream analysis such as Western blotting and RT-qPCR. Immunofluorescent staining revealed nuclear localization of YAP in angiogenic sprouts, a feature previously observed only in 2D cultures. This confirms this model ability to recapitulate pressure-induced YAP activation also in 3D. Our findings support the feasibility of this platform for mechanobiological studies and highlight its potential for investigating angiogenic signalling under controlled pressure conditions. Insight Box A reusable 3D bioreactor enables controlled hydrostatic pressure on endothelial cells within collagen I channels. This model reveals pressure-induced YAP nuclear localization in angiogenic sprouts and supports molecular analysis from single channels, bridging mechanobiology with in vitro vascular modelling.