Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate

Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po₂ levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po₂ ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions.