Mimicking the Liver Microenvironment: 3D Culturing, Organ-on-Chips, and Oxygen Regulation as Strategies to Improve iPSC-Derived Hepatocyte Maturation

As a central organ for metabolic functions, as well as the primary site for the processing of pharmaceuticals and toxins, the liver is frequently modelled in vitro. To achieve more physiologically relevant models that could provide more accurate and predictive results for drug development and disease research, novel approaches such as three-dimensional (3D) cell aggregates, co-culture systems and organ-on-chip (OoC) platforms, have been developed. The breakthrough discovery of somatic cell reprogramming into a pluripotent state in the early 21st century introduced an essentially unlimited cell source, induced pluripotent stem cells (iPSCs), which can be differentiated into other cell types including hepatocyte-like cells (HLCs). This advancement has propelled liver research forward by overcoming the scarce availability of primary human hepatocytes (PHH) and bypassing ethical concerns associated with embryonic stem cells (ESCs). However, iPSC-HLCs often exhibit a foetal-like phenotype with reduced functionality and maturity compared to PHHs. In this thesis, I aim to explore how modifying different aspects of the in vitro microenvironment can enhance the differentiation and promote the maturation of iPSC-HLCs. Firstly, our results demonstrate that 3D iPSC-HLC spheroids exhibit enhanced functionality and prolonged culture longevity in comparison to conventional 2D cultures, highlighting the critical role of culture dimensionality in promoting cell-cell interactions and maintaining natural cellular polarisation. To enable 3D on-chip culturing, various biomaterials were evaluated to identify the most suitable hydrogel for supporting iPSC-HLC functionality and culture dimensionality. Based on our gene expression data, albumin secretion results and practical handling considerations, Geltrex and fibrin emerged as suitable candidates. Both materials exhibited minimal gel degradation, fibrin when supplemented with aprotinin, and supported cell viability throughout the culture period. Moreover, incorporating cell types of mesenchymal origin into multilineage on-chip cultures improved iPSC-HLC functionality, as evidenced by elevated expression of liver-specific genes and proteins. Similar improvements in functionality were observed when iPSC-HLCs were cultured on Liver-Lobule-on-Chip (LLoC) chip devices, which incorporate structural in vivo liver microarchitecture with continuous, pump-driven media perfusion. Immunocytochemical staining of LLoC and 2D control cultures revealed pronounced differences in the expression of mature hepatocyte markers, with LLoC cultures exhibiting a more mature phenotype. Notably, zonation-like patterns, resembling the anatomical and functional organisation of hepatocytes in vivo, were observed at the end of the culture period. These results underscore how high physiological mimicry of the culture environment can guide the differentiation of iPSC-HLCs towards more mature phenotype. To mimic the in vivo oxygen conditions of the liver, which exhibit naturally occurring oxygen gradient with much lower oxygen levels compared to atmospheric oxygen levels and standard incubator conditions (19-21% O2), iPSCs were cultured on commercial 1-well platforms enabling us to establish physiologically accurate oxygen (5-10% O2) levels within the iPSC-HLC cultures. Significant increases in liver-specific functions, albumin and urea secretion, were measured, indicating that correctly timed physioxia during the differentiation process can greatly enhance the maturity of iPSC-HLCs. Interestingly, some zone-specific functions were upregulated within the same conditions, indicating generally enhanced hepatic functionality rather than the emergence of zone-specific cell populations. Overall, the findings of this thesis underscore the critical role of physiological mimicry in in vitro liver modelling. Several factors, such as 3D spheroid culture, co-culture systems, microfluidic platforms, media perfusion, and physioxic conditions, were shown to influence iPSC-HLC differentiation efficiency and improve their maturity. Although it is challenging to identify a single most effective modification due to their interrelated nature, the results collectively highlight the importance of integrating these elements to enhance relevance of in vitro liver models and their translatability.