Shift from 2D to 3D in the development of a human skin cell-derived hepatic in vitro model for toxicological applications

Drug-induced liver injury (DILI) is a major threat to human health and is, together with the low efficacy of generated pharmaceuticals, at the basis of a 90% failure rate in drug development. As preclinical data, obtained using experimental animals, do not adequately represent the human situation, performing in vitro models based on human cells are highly needed. In this doctoral thesis, the relevance for toxicological screening of using human skin-derived stem cells that have been differentiated towards hepatic cells (hSKP-HPC) has been highlighted. It could be shown that these cells, cultured as a simple monolayer, are suitable for investigating drug-induced phospholipidosis (DIPL). Upon exposure to amiodarone, known to induce phospholipidosis in humans, hSKP-HPC displayed typical hallmarks of DIPL, including phospholipids accumulation, lamellar body formation and typical changes in gene expression. As hSKP-HPC monolayers show an immature hepatic phenotype, the next objective was to try to enhance their maturation. Changes in intrinsic properties of undifferentiated stem cells in 3D spheroid cultures versus 2D monolayer culture conditions were explored. It was found that the 3D configuration of hSKP spheres plays a pivotal role in the preservation of the stemness signature. Consequently, 3D nano-scaffolds were generated by two-photon polymerization of biocompatible photopolymers to mimic the micro-architecture of hepatic cell plates in human liver. It could be demonstrated that these scaffolds supported homing and proliferation of hSKP. Overall, these findings emphasize the biological importance of ‘in vivo-like’ conditions for culturing and differentiating human stem cells. Although further optimization is necessary, our results further pave the way to enhance hepatic maturation which is a prerequisite to reliably predict human-specific hepatotoxicity of new pharmaceuticals.