Kristin Beirow, Christian Schmidt, Britta Jürgen, Rabea Schlüter, Thomas Schweder, Patrick J. Bednarski

• The immortalized mouse liver cell line TAMH has been described as a valuable tool for studying hepatotoxic mechanisms, but until now, it has only been reported to grow as a monolayer in culture. However, culturing hepatocytes as three-dimensional (3D) spheroids has been shown to result in improved liver-specific functions (e.g., metabolic capacity) by better mimicking the in vivo environment. This approach may lead to more reliable detection of drug-induced liver injury (DILI) in the early phase of drug discovery, preventing post-marketing drug withdrawals. Here, we investigated the cultivation of TAMH as 3D spheroids, characterizing them with optical and transmission electron microscopy as well as analyzing their gene expression at mRNA level (especially drug-metabolizing enzymes) compared to TAMH monolayer. In addition, comparisons were made with spheroids grown from the human hepatoblastoma cell line HepG2, another current spheroid model. The results indicate that TAMH spheroids express hepatic structures and show elevated levels of some of the key phase I and II drug-metabolizing enzymes, in contrast to TAMH monolayer. The in vitro hepatotoxic potencies of the drugs acetaminophen and flupirtine maleate were found to be very similar between TAMH spheroidal and the monolayer cultures. Both the advantages and disadvantages of TAMH spheroids as an in vitro hepatotoxicity model compared to monolayer model are discussed.