Spontaneous formation of highly functional three-dimensional multilayer from human hepatoma Hep G2 cells cultured on an oxygen-permeable polydimethylsiloxane membrane.

When considering high-density liver cell cultures, adequate delivery of oxygen to the cells appears particularly crucial since it is one of the most limiting parameters of hepatocytes' functions. Here we report on the effects of direct oxygenation through a gas-permeable polydimethylsiloxane membrane on liver-derived cell culture. We used highly proliferative human hepatoma Hep G2 cells to assess the growth-related limitation of such direct oxygen supply. As a consequence of the greater oxygen availability, the proliferation of Hep G2 cells was markedly enhanced, leading to the formation of a thick three-dimensional cellular multilayer. This was supported by the oxygen concentration profiles in the vicinity of the cell layers, predicted by numerical simulations. The applied cells also displayed an increased functionality as assessed by the high-albumin production rates, with per well-based albumin secretion reaching after 15 days 50 times that of the cells cultured in conventional polystyrene plates. Anaerobic glycolysis was in the same time significantly reduced as assessed by both the reduced glucose consumption and lactate production rates. Direct oxygenation through such a gas-permeable membrane appears suitable for engineering functional thick liver tissues for in vivo implantation, as well as for microplate-based chemical/drug screening applications.