Abstract

Predicting drug- and chemical-induced hepatotoxicity remains a great challenge. Animal-based testing during compound development too often fails to identify this risk, unable to mimic the role of non-parenchymal cells in response to compound perturbations and disease progression. Conventional in vitro hepatic model systems, such as monocultures of primary human hepatocytes, lack or exhibit the loss of key functions as well as cellular architecture and integrity over time. We have developed an all-human Tri-Culture System, in both 24- and 96-well formats, comprised of primary human hepatocytes with endothelial and stromal cells derived from donated human tissues. Optimal cell densities and ratios were established based on the restoration and maintenance of key self-assembled cellular structures and functions over a 4-week period, measured by the localization of key cell-cell interaction and junctional polarity markers, albumin and urea synthesis, and CYP basal and induced activities. Experiments comparing mono- and tri-cultures of hepatocytes from multiple donors, healthy and diseased, demonstrated that basal albumin and urea synthesis rates and CYP basal and induced activities decreased markedly (>50% decrease of albumin and urea, undetectable levels of CYP basal expression due to cell loss) in monocultures. These cellular and biochemical parameters increased in the tri-cultures over the initial 4 to 7 days, reaching steady-state levels between 7 and 10 days (albumin and urea expression maintained within a 20% range, and CYP fold change of >10). Regardless of donor background, the presence of non-parenchymal cells sustained and enhanced hepatocyte integrity, polarity and performance over several weeks in culture. Overall, this all-human Tri-Culture System represents an exciting and promising new analytical tool for studying the efficacy and liability of compounds on hepatocellular function of both healthy and diseased human donors.