The gene expression is up-regulated by transfection to spheroids rather than monolayered cells. Monolayered hepatocytes form spheroids on rubbed polyimide membrane after 3 day incubation. The transfection of a rhodamine-labeled plasmid DNA or a green fluorescent protein (GFP) reporter gene to monolayered hepatocytes makes the spheroids exhibiting the fluorescence from the rhodamine or GFP inside the resulting spheroids. On the other hand, the transfection to hepatocyte spheroids makes the spheroids exhibit the fluorescence only outside the resulting spheroids. However, the whole gene expression of the luciferase reporter plasmid DNA from the lysate of the transfected spheroids is up-regulated in early incubation time, as compared to that of the transfected monolayered hepatocytes. Furthermore, the secretion of vascular endothelial growth factor (VEGF) from the VEGF-transfected spheroids is higher than that from the transfected monolayered hepatocytes. These results suggest that the up-regulation of exogenous gene expression is achieved by the control of differentiation and proliferation of hepatocytes via spheroid formation.