Enhanced cytochrome P450IA1 activity of self-assembled rat hepatocyte spheroids

Primary rat hepatocytes can self-assemble to form multicellular spheroids w hen plated onto Primaria petri dishes. Spheroids have been observed to exhi bit enhanced liver-specific functions and differentiated ultrastructure com pared to monolayer cultures on dry collagen. With confocal scanning laser m icroscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. Th is highly fluorescent molecule is the P450-mediated product of 7-ethoxyreso rufin O-dealkylation (EROD). Significantly higher P450 activity was observe d in spheroids compared to monolayers on collagen upon induction with 50 mu M beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measur ing microsomal EROD activity. The distribution of CYP1A1 activity within sp heroids was heterogeneous, with higher activity localized to the hepatocyte s in the interior. During the process of spheroid formation, cells were ini tially seen to attach and spread out as a monolayer. This stage was associa ted with relatively low CYP1A1 activity. As cells formed multicellular stru ctures and aggregated into spheroids, the level of CYP1A1 activity increase d over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The hi gher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model syste m for investigating drug metabolism, tissue engineering, and tissue self-as sembly.