5). We observed that trovafloxacin and APAP induced increased dose- and time-dependent cytotoxicity and decreased cell viability at physiologically relevant drug concentrations close to the Cmax (Table 1) after chronic exposure (8 or 15 days) of human 3D liver cultures, but not after acute treatment (2 days) of human 2D

hepatocyte monolayers (Figs. 5A and B). Almost no toxicity was detected with levofloxacin and AMAP in 3D liver cells and 2D hepatocytes (Figs. 5A and B). As described above, troglitazone elicited cytotoxicity and cell viability INK 128 in vivo decrease in human but not in rat 3D liver cells (Fig. 4B). To further characterize this effect, we measured additional endpoints after repeated drug-treatments (up to 8 days) including cell apoptosis (caspase 3/7 activity), cell cytotoxicity (dead-cell protease activity and ALT-release) and cell viability (live-cell protease activity). In human 3D liver cells, troglitazone elicited

cytotoxicity (LDH release) already after 1 day of treatment, demonstrating an acute drug effect (Fig. 6A). After 8 days of treatment, trogitazone caused a dose-dependent increase in caspase 3/7 and dead-cell protease activity, as well as decrease in live-cell protease activity in human 3D liver cells (Fig. 6A). In contrast, pioglitazone applied during 8 days to human 3D liver cells Astemizole did not cause any significant changes in TGF-beta inhibitor any of these parameters (Fig. 6B). In agreement with this result, we observed a dose-dependent increase of ALT release in troglitazone, but not in pioglitazone treated human 3D liver cells (Figs. 6A and B). In this work, we characterized a 3D liver culture model of rat and human for detection of single or repeated drug-treatment induced toxicities. We compared the 3D liver co-culture model with standard monolayer hepatocytes grown on collagen type I, since this

model is one of the most frequently used model in pharmacological industry for drug toxicity screening and mechanistic studies. Other culture models expose hepatocytes between two layers of Matrigel and/or collagen-I gels that allow better retention of cell cyto-architecture, CYP activity and prolonged functional lifespan for up to 14 days compared with cultures on rigid collagen (Dunn et al., 1989, Guguen-Guillouzo and Guillouzo, 2010, Hewitt et al., 2007, LeCluyse, 2001, Lecluyse et al., 2012 and Mingoia et al., 2007). Recent data have shown that heterotypic cell–cell interactions between parenchymal and nonparenchymal cells induce higher levels of phenotypic functions in human hepatocytes than extracellular matrix configuration or composition (Griffith and Swartz, 2006, Guillouzo, 1998 and LeCluyse, 2001).