To optimize the dimensions of the gradient chamber, a coupled convection-diffusion-kinetic model with oxygen as the limiting substrate was developed. The model was used to describe hepatocyte proliferation and metabolism in the gradient chamber. Kinetic parameters were obtained from the literature for human hepatocarcinoma (HepG2) cells. Simulation results predicted that the oxygen tension could be maintained above the effective Km for a culture compartment volume of up to 4.8x10^-4 cm^3 by distributing the medium inlets along the length of the reactor. The maximum attainable density was 10^5 cells.
Based on the simulation results, gradient chambers were fabricated using standard soft-lithography techniques and PDMS molding. To maintain consistency with the mathematical model, the HepG2 cell line was used as the model cell system. Cultures remained viable and exhibited hepatocyte metabolic function for at least 10 days. On day 7 after seeding, cultures were treated with the test drug troglitozone (TGZ), an insulin sensitizing, anti-diabetic compound first approved for clinical use, but eventually withdrawn due to indications of idiosyncratic ADR. A linear gradient of the drug was applied. Viability and metabolic assessments using fluorescent probes estimated that TGZ causes significant toxicity above 100 uM, in good agreement with conventional well-plate assay results. Taken together, our results to date indicate that the gradient chamber is an efficient platform for in vitro drug toxicity studies.