Long‐Term Stability of Primary Rat Hepatocytes in Micropatterned Cocultures

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac™). Here, we assess the long-term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96-well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel™ sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW-7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3-hydroxy-3-methylglutaryl-CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96-well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways.