Xenobiotic Metabolism in Alginate-Encapsulated Primary Human Hepatocytes Over Long Timeframes

Abstract

Introduction: Application of in vitro-to-in vivo extrapolation to chemical safety assessment has gained significant attention in the past few years. Accurate prediction of hepatic metabolic clearance from in vitro data is critical in this regard as it determines compound pharmacokinetics and exposure at sites of action. To estimate hepatic metabolic clearance, intrinsic clearance (CLint) is typically measured using in vitro systems such as primary hepatocytes or subcellular fractions derived from liver tissues. However, currently available in vitro systems used in these studies often lack long-term metabolic competence, making it challenging to estimate CLint for poorly metabolized and slowly cleared chemicals. Materials and Methods: To address this challenge, we developed a three-dimensional (3D) primary human hepatocyte cell culture system using alginate hydrogels (“alginate bead culture”) with extended viability and metabolic competence. Results: Alginate bead culture generates comparable estimates of metabolism to other in vitro and in vivo systems. In addition, expression of several xenobiotic metabolizing enzymes over long timeframes in beads is comparable to freshly isolated hepatocytes in suspension culture. Discussion: We compare CLint estimated from traditional suspension versus alginate bead culture for two chemicals (S-warfarin and coumarin) and show that alginate bead culture holds promise to increase confidence in estimates of CLint for slowly cleared chemicals. Conclusions: In addition to providing an improved in vitro tool to estimate metabolic clearance for a broad range of chemicals, this simple 3D hepatocyte culture can be adapted to a continuous flow-based bioreactor system allowing repeated exposure experiments over long periods in vitro, tackling an important challenge in in vitro-based chemical safety assessment.