In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Liver sinusoidal endothelial cells (SECs) are generally refractory to extended in vitro culture. In an attempt to recreate some features of the complex set of cues arising from the liver parenchyma, we cocultured adult rat liver SECs, identified by the expression of the marker SE-1, with primary adult rat hepatocytes in a 3D culture system that provides controlled microscale perfusion through the tissue mass. The culture was established in a medium containing serum and VEGF, and these factors were then removed to assess whether cells with the SE-1 phenotype could be supported by the local microenvironment in vitro. Rats expressing enhanced green fluorescent protein (EGFP) in all liver cells were used for isolation of the SE-1-positive cells added to cocultures. By the 13th day of culture, EGFP-expressing cells had largely disappeared from 2D control cultures but exhibited moderate proliferation in 3D perfused cultures. SE-1-positive cells were present in 3D cocultures after 13 days, and these cultures also contained Kupffer cells, stellate cells, and CD31-expressing endothelial cells. Global transcriptional profiling did not reveal profound changes between 2D and 3D cultures in expression of most canonical angiogenic factors but suggested changes in several pathways related to endothelial cell function.

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