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Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher 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 system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Xenogeneic hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, from multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 microns. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Urea-genesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.