Reconstructing physiological oxygen gradients reveals the role of hypoxia in colon epithelial organization

Oxygen gradients organize tissue architecture and metabolism ^1,2 , yet their precise spatial profiles and mechanistic roles remain poorly understood because both <i>in vivo</i> measurement and <i>in vitro</i> control are technically challenging ^3,4 . Here, we quantify the oxygen landscape of the mammalian intestine using microscale sensors, revealing a steep luminal-basal gradient of approximately 10-60 µM mm ^- ¹ that collapses under antibiotic perturbation. We then recreate this physiological range <i>ex vivo</i> with a submerged chemostat microfluidic platform that fixes the oxygen boundary condition by coupling an oxygen-permeable PDMS chip to an external scavenger reservoir and integrating embedded optical sensors for real-time readout. This architecture suppresses ambient oxygen ingress and sustains programmable gradients of 10-20 µM mm ^- ¹ across three-dimensional colorectal cancer organoid cultures while remaining compatible with live imaging and endpoint retrieval. The platform bridges quantitative <i>in vivo</i> oxygen mapping with controlled <i>ex vivo</i> modeling, establishing a generalizable approach to interrogate how spatial oxygen dynamics govern epithelial organization and disease progression.