Genomic Approaches to self-assembly and drug metabolism in spheroids

Cultured primary hepatocytes are widely used for drug metabolism and toxicity studies in vitro. Primary hepatocytes in culture typically lose their viability in about a week. When cultured in 3-D aggregates called spheroids, hepatocytes are not only able to survive in culture for extended periods of time (more than 4 weeks), but also express enhanced liver-specific functions such as albumin synthesis, ureagenesis and cytochrome P450 activity as compared to monolayers.

To understand the molecular basis of extended viability and function in hepatocyte spheroids, suppression subtractive hybridization (SSH) and cDNA microarray and quantitative PCR are being used to compare gene expression patterns at various stages of porcine hepatocyte spheroid formation in suspension culture. The long viability in culture and enhanced liver functions make spheroids ideal for in vitro studies for drug screening and investigating the long-term effects of xenobiotics. For drug metabolic studies, we employ porcine hepatocytes.

Pigs are very similar to humans at the physiological, anatomical and sequence level. Hence, porcine hepatocyte spheroids may form an ideal platform for large-scale drug screening applications using genomic tools. To explore the global response to drug treatment in porcine spheroids, we used human cDNA microarrays from Agilent to screen for differentially expressed genes upon induction with Phenobarbital (PB). Many genes related to the drug metabolic pathway, including cytochrome P450s, UGTs, retinaldehyde dehydrogenase, transporters and stress-related genes and antiapoptotic sequences were upregulated in PB-treated spheroids. These results were confirmed by quantitative RT-PCR analysis of the porcine CYP2B and 3A sequences.

Publication:

Narayanan, R.A., Rink, A., Beattie, C.W. and Hu, W.-S. (2002) Differential gene expression analysis during porcine hepatocyte spheroid formation. Mamm. Genom. 13: 515-523.

Team Members:

R.A. Narayanan (U of M), Shen Dong (U of M)

Collaborators:

Anette Rink (University of Nevada at Reno), Craig Beattie (University of Nevada at Reno)