Regions of hypoxia are common in solid tumors and are associated with enhanced malignancy, metastasis, and chemo/radio resistance. Real-time hypoxic cellular experimentation is challenging due to the constant need for oxygen control. Most microfluidic platforms developed thus far for hypoxic cell studies are burdened by complex design parameters and are difficult to use for uninitiated investigators. However, open-well microfluidic platforms enable short and long term hypoxic cell studies with an ease of use workflow. Specifically, open-well platforms enable manipulation and addition of cells, media, and reagents using a micropipette for hypoxic cell studies in tunable dissolved oxygen concentrations as low 0.3 mg/l. We analyzed design considerations for open-well microfluidic platforms such as media height, membrane thickness, and impermeable barriers to determine their effects on the amount of dissolved oxygen within the platform. The oxygen concentration was determined by experimental measurements and computational simulations. To examine cell behavior under controlled oxygen conditions, hypoxia-induced changes to hypoxia inducible factor activity and the mitochondrial redox environment were studied. A fluorescent reporter construct was used to monitor the stabilization of hypoxia inducible factors 1α and 2α throughout chronic hypoxia. Reporter construct fluorescence intensity inversely correlated with dissolved oxygen in the medium, as expected. Additionally, the glutathione redox poise of the mitochondrial matrix in living cancer cells was monitored throughout acute hypoxia with a genetically encoded redox probe and was observed to undergo a reductive response to hypoxia. Overall, these studies validate an easy to use open-well platform suitable for studying complex cell behaviors in hypoxia.
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