1A). The cells were equally distributed Daporinad order over the scaffold areas forming a dense tissue. Once the 3D tissue was formed correctly, no microscopic changes were found in the upper layers of cells over time of culture up to 3 months. Cultures which have shown big areas with no or less cells over the scaffold areas were not used for the experiments. To quantitatively assess the stability of liver specific functions of the cells in culture we measured secretion of albumin, transferrin and fibrinogen as well

as urea synthesis, a marker of nitrogen metabolism (Fig. 1B, and Supplementary Fig. 1A). Albumin secretion in human and rat 3D liver cells was stable as from day 12 onwards and remained constant for up to 3 months in culture at a level of 2–3 μg/day/106 hepatocytes. Transferrin secretion in human 3D liver cells reached maximum levels of 5 μg/day/106 hepatocytes at day 34, then slowly decreased until day 77 (Fig. 1B), whereas transferrin secretion in rat 3D liver cells was constant between 2 and 3 μg/day/106 hepatocytes over 90 days in culture (Supplementary check details Fig. 1A). Fibrinogen secretion in human and rat 3D liver cells reached a peak of 4.5 or 7 μg/day/106 hepatocytes at day 15,

then declined and remained constant until the end of the investigated period (Fig. 1B and Supplementary Fig. 1A). Urea synthesis in human 3D liver cells was stable over the Florfenicol entire culture period and reached 250 μg/day/106 hepatocytes. In rat 3D liver cells urea synthesis declined with time from 250 to 150 μg/day/106 hepatocytes (Supplementary Fig. 1A). In contrast to 3D liver cells, primary human and rat hepatocytes grown as a 2D monolayer lost their morphological features and liver specific functions after only a few days (Fig. 1B and Supplementary Fig. 1A, (Guguen-Guillouzo and Guillouzo, 2010, Guillouzo, 1998 and Hewitt et al., 2007). Moreover,

human 3D liver cells had higher levels of albumin-, transferrin- and fibrinogen-secretion and urea synthesis compared to human 2D hepatocytes (Fig. 1B). Rat 3D liver cells had similar levels of albumin- and transferrin-secretion or urea-synthesis as rat 2D hepatocytes. In 2D hepatocyte cultures, all these liver-specific parameters rapidly declined after 3–4 days (Supplementary Fig. 1A). Overall, 3D liver tissues retained liver-specific function for up to 3 months. To assess metabolic competence of human and rat 3D liver co-cultures, we measured basal, inducible and inhibited CYP3A4, CYP3A1/2, CYP1A1 and CYP2C9 activities. CYP activities were measured after treatment of human and rat 3D liver co-cultures for 3 days with vehicle (DMSO), CYP-inducers or CYP-inducers in combination with CYP-inhibitors (Fig. 1C and Supplementary Fig. 1B). We found that human 3D liver cells stably retained basal, inducible and inhibited CYP3A4, CYP1A1 and CYP2C9 activities up to 3 months in culture (Fig. 1B).