Among the features of in vivo liver cells that are rarely mimicked in vitro, especially in microchips, is the very high cell density. In this study, we have cultured HepG2 in a plate-type PDMS scaffold with a three-dimensional ordered microstructure optimally designed to allow cells to attach at a density of 10(8) cells/mL. After the first step of static open culture, the scaffold was sealed to simulate the in vivo oxygen supply, which is supplied only through the perfusion of medium. The oxygen consumption rate at various flow rates was measured. An average maximal cellular oxygen consumption rate of 3.4 x 10(-17) mol/s/cell was found, which is much lower than previously reported values for hepatocytes. Nevertheless, the oxygen concentration in the bulk stream was not the limiting factor. It has been further confirmed by the reported numerical model that the mass transport resistance on the surface of a cell that limits the oxygen supply to the cell. These results further emphasize that access to a sufficient quantity of oxygen, especially through the diffusion-limited layer on the surface of a cell, is very important for the metabolism of hepatocytes at such a high density.