Three-dimensional cell-culture systems using nanofibrillated bacterial cellulose restores drug metabolism activity in HepG2 hepatocellular carcinoma cells

Abstract Three-dimensional (3D) culture more faithfully reproduces in vivo –like cell interactions and functions than conventional two-dimensional (2D) monolayers. Although HepG2 cells are widely used in drug discovery, their 2D cultures exhibit low expression of drug-metabolizing enzymes such as cytochrome P450s (CYPs), limiting utility for toxicity and pharmacokinetic studies. Nano-fibrillated bacterial cellulose (NFBC) is a unique biomaterial that features exceptional homogeneity, high purity, and excellent biocompatibility. We recently employed two NFBC-based 3D culture systems: the Suspension and the OnGel methods. However, the capacity of NFBC to improve hepatocyte-specific functions has yet to be systematically explored. In this study, we aimed to establish a 3D culture platform for HepG2 cells using NFBC to restore hepatic functionality, and drug-metabolizing activity in particular, via comparison with conventional 2D monolayers. Both of these 3D culture systems produced viable HepG2 spheroids with good proliferation. Exploratory microarray profiling suggested broad upregulation of multiple absorption, distribution, metabolism and excretion (ADME) genes in the HepG2 spheroids. Among these, the enzyme Cytochrome P450 3A4 (CYP3A4) showed a significant increase in protein expression and in enzymatic activity in the HepG2 spheroids, which was functionally confirmed by acetaminophen (APAP) toxicity via its bioactivation into toxic metabolite. In addition, the HepG2 spheroids displayed reduced sensitivity to the anticancer drug doxorubicin compared to 2D monolayer. Collectively, these findings demonstrate that NFBC-based 3D culture systems effectively restore liver-specific functions in HepG2 cells, which results in HepG2 spheroids with metabolic competence and physiologically relevant drug responses, and could offer a promising tool for high-throughput in vitro drug screening.