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Tissue engineering within three-dimensional scaffolds represents a developing field with the potential to create in vitro livers. Human liver cells, HepG2, respond to cues in their immediate microenvironment by modulating cell functions including cell proliferation, urea synthesis and drug metabolism. The importance of three-dimensional environments draws from the fact that it has been extensively used for guided tissue regeneration. HepG2 cells grown in traditional two-dimensional supports were compared to three-dimensional cultures in polystyrene scaffolds. The 5-day experimental study examined the effects of the different environments on HepG2 cell urea synthesis using a quantitative colorimetric assay and drug metabolism-specific function using the cytochrome P450 monooxygenase substrate, 7-ethoxy-4-trifluoromethyl-coumarin. The function of liver cells grown on three-dimensional scaffolds was enhanced compared to the activity of cells grown on two-dimensional supports. The results suggest that HepG2 liver cell function in three-dimensional environments more closely mimic physiological responses than existing two-dimensional culture systems.