Oxygen gradient and humidity play a crucial role in long-term cell culture within the multilayer microfluidic devices. This paper reports an integrated chip for long-term cell culture with controllable humidity and dissolved oxygen levels, consisting of a gas layer, a water jacket layer, a control layer and a culture layer. Parallel gas mixing channels are utilized to precisely regulate dissolved oxygen concentrations above the culture chambers from 0 ppm to 42 ppm. In addition, numerical analysis is implemented to investigate humidity levels around the culture zones where the water jacket layer is assembled under the gas layer. The analysis shows that the region around each chamber is nearly saturated. To validate the applicability of the device for long-term bacteria cell culture with various oxygen concentrations,, three sets of experiments are conducted with different bacteria (Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum). These also demonstrate the potential applications in dynamic monitoring of cell growth and cell-cell interactions.
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