A 3D-printed multi-channel microfluidic device for precise dissolved oxygen regulation in cancer hypoxia research

Hypoxia plays a critical role in cancer progression, therapy resistance, and metastasis, yet establishing precise, physiologically relevant dissolved oxygen (DO) gradients for in vitro cell studies remains technically challenging. Here, we developed a novel 3D-printed microfluidic platform that generates eight parallel DO gradients (0-100 % saturation) for multiplexed hypoxia research. The integrated system combines a modular microplate adapter with a precise DO concentration gradient generation chip, creating stable linear oxygen profiles that faithfully mimic different in vivo tumor microenvironments. DO concentration gradient generation is validated with online electrochemical detection showed great linearity (R² > 0.99) and accuracy. The microfluidic perfusion system demonstrated excellent biocompatibility, supporting robust cellular proliferation and maintaining >95 % viability across all cell lines during 72-h continuous culture periods. Using renal (A498) and colorectal (SW480/SW620) cancer cell lines, we demonstrate concentration-dependent cellular responses to hypoxia. Compared to conventional microplates and microfluidic chips, this system enables stable perfusion culture of cells, and high-resolution analysis of spatiotemporal hypoxia adaptations. This technology provides a versatile, cost-effective tool for investigating hypoxia-driven mechanisms in cancer biology and therapeutic development.