An integrated microfluidic culture model for photodynamic therapy evaluations under normoxic and hypoxic conditions

Photodynamic therapy (PDT) is a clinically approved cancer treatment that combines photosensitizer, oxygen, and light to generate cytotoxic reactive oxygen species (ROS). Its efficacy is often compromised by restricted drug and light penetration, and hypoxic microenvironments in solid tumors, making it essential to establish in vitro models that can recapitulate these features for PDT studies and treatment optimization. In this work, we present a microfluidic platform incorporating an agarose gel-embedded tumor cell culture that enables precise geometric and environmental control, including defined microchamber architecture, dynamic perfusion-based photosensitizer delivery, oxygen regulation, and tunable light exposure for systematic PDT efficacy evaluation. Computational fluid dynamics (CFD) and discrete element method (DEM) simulations guided the optimization of loading agarose-cell suspensions into confined microchambers. The hexagonal microchamber layout and hydrogel matrix supported cell positioning within controlled diffusion domains. A modular dye-based optical filter system generated spatially tunable light fluence, while integrated gas control regulated normoxic and hypoxic conditions. PDT studies revealed that dynamic perfusion and microenvironmental constraints produced a spatial gradient of treatment response along the flow direction of photosensitizer. ROS generation was markedly higher under normoxia than hypoxia, and live/dead and CCK-8 assays confirmed a light dose-dependent decline in viability under normoxia, whereas hypoxic cultures maintained > 85% viability. Collectively, by capturing key tumor microenvironmental features such as diffusion limitations and oxygen deprivation, this platform offers a useful tool for photosensitizer assessment, PDT protocol optimization, and mechanistic investigation of oxygen-dependent PDT responses.