Objective

Drug metabolism by liver enzymes and toxicity of drug candidates to the liver are important considerations to ADME and drug safety testing. Novel 3D culture techniques that can maintain or enhance in vitro functionality of model liver cell lines can help streamline the drug discovery pipeline and increase confidence in drug safety prior to clinical trials. We used 96-well plate Alvetex Scaffold for the culture of HepG2 cells, to create a high-throughput in vitro 3D model for drug hepatotoxicity testing. HepG2 cells were cultured in Alvetex Scaffold 96-well plates for 7 days prior to addition of a panel of known hepatosafe (Ibuprofen, Entacapone, Rosiglitazone) and hepatotoxic (Ibufenac, Tolcapone, Troglitazone, Acetaminophen, Gemfibrozil) compounds. Cell viability was assessed after 7 days (MTS assay) and EC90 values were compared to clinical CMax values reported in the literature, demonstrating 100% specificity and 60% sensitivity. HepG2 cells were also cultured for up to 28 days in the absence of drug compounds to test the model's potential use for long-term repeat drug dosing and showed stable cell viability throughout. Secretion of urea, albumin and lactic acid in the culture medium was detected at similar levels at both 7 and 14 days, and liver enzyme activity was suggested by substrate degradation of Midazolam (CYP3A4 substrate), Diclofenac (CYP2C9 substrate) and 7-OH-Coumarin (phase II enzyme substrate), as assessed by LC-MS-MS.