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 in vivo measurement and in vitro 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 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 µM 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.