Emulsion Templated Porous Polymers as Scaffolds for 3D Hepatocyte Culture

Abstract

Hepatocytes are the main functional cells of the liver and are used extensively in vitro for predicting in vivo drug toxicity profiles. However, the predictive accuracy of in vitro hepatocyte models depends on the physiological relevance of the artificial growth environment. Conventional in vitro hepatocyte models have employed monolayer cultures on two-dimensional (2D) substrates, forcing cells into a flattened morphology that is far removed from the in vivo scenario. Unsurprisingly, 2D cultures often show significant deviations from native liver genotype and phenotype and so are unable to accurately predict drug toxicity. Accordingly, it is hypothesised that approximating the native liver three-dimensional (3D) tissue architecture in vitro will help to preserve genotype and phenotype and so improve predictive accuracy.

In this study, emulsion templated porous polymers were investigated as scaffolds for 3D hepatocyte culture. In particular, porous polystyrene scaffolds were explored due to their high porosity, reproducibility and suitable mechanical strength properties. Hepatocytes were cultured on polystyrene scaffolds under a range of culture conditions and were found to approximate native liver density and architecture. The morphology of hepatocytes in scaffolds was representative of in vivo, unlike the flattened morphology of 2D cultures. Crucial ultrastructural features involved in drug detoxification such as bile canaliculi were also present in scaffold cultures, but almost absent from 2D cultures. Importantly, these representative structural features translated into functional and genetic improvements in vitro. Hepatocytes in scaffolds displayed increased albumin synthesis, a key marker of hepatocyte function. Hepatic cell lines also showed increased resistance to drug toxicity compared to 2D cultures. Hepatic drug metabolising genotype was also increased to more physiologically relevant levels in scaffolds compared to 2D cultures.

In addition, emulsion templated polystyrene scaffolds were also made more biochemically relevant by surface functionalising with galactose, a ligand known to selectively bind to hepatocytes in vivo via the asialoglycoprotein receptor (ASGP-R). Scaffold morphology was maintained with the incorporation of galactose, allowing cells to approximate native liver tissue architecture. Moreover, the pendent galactose ligands were found to be accessible to hepatocytes adhering onto the scaffold.

In summary, this thesis has shown that emulsion templated porous polymers can offer a more physiologically relevant growth environment for hepatocytes in vitro. This could have a profound effect on improving drug toxicity predictions and so reducing the dependence on animal testing.