Three-dimensional culture of hepatocytes in a continuously flowing medium

Rat hepatocytes were maintained on three-dimensional cultures on sponge discs kept in Spinner Baskets (New Brunswick Scientific Co., New Brunswick, NJ, USA) with continuously circulating serum-free hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Hepatocytes were embedded in polyester sponge discs with a collagen gel at the concentration of 5 million cells/ml. Atmospheric gas containing 7% CO2 was directly bubbled into the medium. Agitation by the impeller created a continuous medium-flow through the packed hepatocytes. Comparison between identically prepared perfused and stationery cultures showed that hepatocytes in the perfused cultures maintain higher levels of DNA synthesis. These results demonstrate the value of perfusion systems and also show that hepatocytes can proliferate and maintain differentiation in three-dimensional culture environments.