1B and Supplementary Fig. 1A). Our results are in agreement with published data on 3D liver model created by RegeneMed demonstrating that a 3D architecture allows rat liver cells to maintain secretion of liver specific proteins for up to 48 days in culture (Naughton et al., 1994 and Naughton et al., 1995). In humans, around 12 g albumin is synthesized and secreted per day in a 70 kg man, which corresponds to 60 μg secreted albumin/106 hepatocytes/day (Khalil et al., 2001).

Human 3D liver cells secrete lower amounts of albumin than the human APO866 molecular weight liver but 4 to 6 times higher levels than the 2D hepatocytes (Fig. 1B). The normal human plasma concentration of fibrinogen is 1.5–3.5 g/l and of transferrin is 2.3–3.9 g/l with half-life of 4 days (Acharya and Dimichele, 2008) and 8 days (Bates and McClain, 1981), respectively. This corresponds to 6–14 μg/106 hepatocytes/day of fibrinogen and 5.2–8.78 μg/106 hepatocytes/day Inhibitor Library solubility dmso of transferrin secreted by human liver in vivo. Thus, the levels of secreted fibrinogen and transferrin by human 3D liver cells ( Fig. 1B) are comparable with the in vivo situation. The amount of urea synthesized by human liver is 181.8 μg/106 hepatocytes/day ( Khalil et al., 2001 and Rudman et al., 1973) similarly to the amount of the urea synthesized by the human 3D liver model ( Fig. 1B). Variability in hepatocyte viabilities and platability as well

as liver specific function were observed between cells from different donors. For the creation of the 3D liver co-cultures were used hepatocytes which have cell viability above 80% and good adhesion capacity to the scaffold as assessed by visual inspection 2 days after seeding. To obtain as consistent results as possible, for the liver functionality and the drug-toxicity studies only those cultures which met the following criteria were taken for further experimentation: scaffold completely covered with cells (only few detached cells in first

medium removal), levels of albumin ≥ 1 μg/day/million hepatocytes and inducible CYP3A4 activity. The presence of endothelial, Kupffer and hepatic stellate cells together with ECM components and the preserved 3D cell architecture has been shown to prolong the survival of hepatocytes Pyruvate dehydrogenase and to improve their function by increasing the secretion of albumin and induction of CYP activity (Begue et al., 1983, Dash et al., 2009, Khetani and Bhatia, 2008, Kuri-Harcuch and Mendoza-Figueroa, 1989, Michalopoulos et al., 1979, Peterson and Renton, 1984 and Yuasa et al., 1993). We have shown that the secretion of liver specific proteins in human and rat 2D hepatocytes declined after only a few days in culture (Fig. 1B and Supplementary Fig. 1A), which is also in line with the previous published results (Guguen-Guillouzo and Guillouzo, 2010, Hewitt et al., 2007 and Lecluyse et al., 2012).