Abstract
Liver zonation is a fundamental characteristic of hepatocyte spatial heterogeneity, which is challenging to recapitulate in traditional cell cultures. This study presents a novel microfluidic device designed to induce zonation in liver cell cultures by establishing an oxygen gradient using standard laboratory gases. The device consists of two layers; a bottom layer containing a gas channel network that delivers high (cell incubator air, 19% oxygen) and low oxygenated (nitrogen) gases to create three distinct zones within the cell culture chamber in the layer above. Computational simulations and ratiometric oxygen sensing were employed to validate the oxygen gradient, demonstrating that stable oxygen levels were achieved within two hours. Liver zonation was confirmed using immunofluorescence staining, which showed zonated albumin production in HepG2 cells directly correlating with oxygen levels and mimicking in-vivo zonation behavior. This user-friendly device supports studies on liver zonation and related metabolic disease mechanisms in vitro. It can also be utilized for experiments that necessitate precise gas concentration gradients, such as hypoxia-related research areas focused on angiogenesis and cancer development.