Improving hepatic metabolic function with 3D iPSC-derived human hepatocytes

Human induced pluripotent stem cells (iPSCs) have the potential to significantly advance drug discovery and clinical applications, particularly in personalized medicine. Synthetic biocompatible materials improve the reproducibility and efficiency of in vitro protocols and enable three-dimensional (3D) cell culture. 3D spheroids and organoids are known to promote hepatic maturation and improve overall liver function. In this study, 3D hepatocyte-like cells (3D HLCs) were differentiated from human iPSCs using a synthetic polypeptide hydrogel scaffold. Their metabolic capacity, including drug biotransformation and mitochondrial function, was assessed using LC-MS semi-targeted metabolomics, providing functional characterization beyond marker expression levels. Compared with conventional monolayer (2D) cultures, 3D HLCs displayed a more mature hepatic phenotype. They acquired xenobiotic metabolism capacity earlier in differentiation, at day 30 rather than day 40 in 2D HLCs. In addition, 3D HLCs showed increased CYP3A4 activity (> 20-fold) and CYP2D6 activity (2-11-fold), as well as enhanced mitochondrial function, including higher β-oxidation, relative to 2D HLCs. The cytotoxic effects of eight drug-induced liver injury (DILI) compounds-amiodarone, azathioprine, cyclosporine A, diclofenac, nefazodone, omeprazole, rosiglitazone, and troglitazone-were also evaluated in 2D and 3D HLCs and compared with 2D primary human hepatocytes (2D PHHs). Drug exposure caused lower cell viability in 3D than in 2D HLCs, more closely resembling the response observed in 2D PHHs. Overall, this study demonstrates the applicability of 3D HLCs for metabolism and toxicology studies, with potential value for personalized drug testing and disease modeling.