Advanced In Vitro Systems for Studies of Drug Disposition in the Human Liver

In drug development, in vitro models are used to assess specific aspects of in vivo Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of the drugs. Relevant in vitro assays play a crucial role in bringing safe and efficacious compounds to the market, and contribute to the Replacement, Refinement and Reduction (3Rs) of animal experiments. Much effort is now being directed to the development of different physiologically relevant advanced in vitro models. One of such models is three-dimensional spheroids of primary human hepatocytes (3D PHH). These 3D PHH closely resemble the in vivo liver at the transcriptome, proteome and metabolome levels. However, 3D PHH are cultured under different conditions and the reproducibility of these culture varies greatly across laboratories. This thesis contributes to harmonization of 3D PHH culture approaches. First, the effect of the cell culture medium on 3D PHH was evaluated. We compared various commercially available media with undisclosed or known content, and also assessed the influence of commonly used medium components such as glucose, insulin, zinc and foetal bovine serum. The choice of cell culture medium had a pronounced effect on the hepatic phenotypes. Importantly, we demonstrate that 3D PHH could be successfully cultured in the animal-serum free physiologically relevant medium with fasting levels of insulin and glucose. Further, we appraised the effect of ultra-low attachment culture plates on the performance of 3D PHH, and demonstrated that Corning and Biofloat plates facilitate the formation of spheroids with most physiologically relevant phenotypes. Throughout all projects included in this doctoral thesis, mass-spectrometry based global proteomics served as indispensable tool for phenotypic description of 3D PHH. However, the choice of workflow for this analysis has a significant impact on biological interpretation. Here, twelve different proteomics workflows for phenotypic description of 3D PHH were compared, and these results will aid researcher in our field in making an informed decision on the approach to the phenotypical screening of liver spheroid cultures. In conclusion, this thesis provides an improved understanding and optimization of 3D primary human hepatocyte spheroid cultures, and deep integration of this in vitro model into drug development pipelines.