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

Oxygen gradients organize tissue architecture and metabolism1,2, yet their precise spatial profiles and mechanistic roles remain poorly understood because both in vivo measurement and in vitro control are technically challenging3,4. Here, we quantify the oxygen landscape of the mammalian intestine using microscale sensors, revealing a steep luminal-basal gradient of approximately 10-60 {micro}M mm-1 that collapses under antibiotic perturbation. We then recreate this physiological range ex vivo 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 {micro}M mm-1 across three-dimensional colorectal cancer organoid cultures while remaining compatible with live imaging and endpoint retrieval. The platform bridges quantitative in vivo oxygen mapping with controlled ex vivo modeling, establishing a generalizable approach to interrogate how spatial oxygen dynamics govern epithelial organization and disease progression.