Non-alcoholic fatty liver disease (NAFLD) is a family of liver diseases associated with obesity. Initial stage of NAFLD is characterized by a fatty liver, referred to as steatosis, which progresses in some individuals to nonalcoholic steatohepatitis (NASH) and liver failure. In order to study and treat the many liver diseases such as NAFLD, an improved in vitro cellular model is needed. Several studies in the past have attempted to elucidate these mechanisms using primary hepatocytes or relevant hepatoma cell lines in two-dimensional (2D) monolayer in vitro cultures. These 2D planar culture systems, unfortunately, do not represent the complex architecture of hepatic tissue in vivo. Therefore, we have engineered an elastin-like polypeptide (ELP)-polyethyleneimine (PEI) copolymer and shown that ELP-PEI coated surfaces influenced H35 rat hepatoma cell morphology to create 3D spheroids. Our reporter cell model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, decreased proliferation, decreased liver-specific function, and increased reactive oxygen species accumulation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor kappa B (NF kappa B) in response to its classical inducer (tumor necrosis factor-alpha, TNF-alpha) under lean and fatty conditions in both 2D and 3D culture configurations. We found that, in 3D spheroids, linoleic acid treatment activated NF kappa B at earlier time points during the development of steatosis, but suppressed the TNF-mediated NF kappa B activation at later time points. These studies therefore provide a good starting point to evaluate such relationships observed during NAFLD in a 3D in vitro cell culture. Biotechnol. Bioeng. 2011; 108: 1171-1180. (C) 2010 Wiley Periodicals, Inc.