V9002

Various groups of cell culture models were used in our study that included: 2D, cells were cultured in a dish in a routine two-dimensional manner; 3D, cells were encapsulated and cultured under static conditions; and 3D-F, cells were encapsulated and cultured in the fluidized bed bioreactor. CYP450 1A2, 3A4, 1A1 and 1B1 enzyme activity assays were carried out directly in 24-well plates. The measurement of luciferase activity was performed with a P450-Glo CYP1A2 assay (V8422), a CYP3A4 assay (V9002), a CYP1A1 assay (V8752) and a CYP1B1 assay (V8762) (all from Promega, Madison, WI, USA). In brief, the cells were incubated at 37°C in Krebs-Henseleit buffer (K3753; Sigma-Aldrich, St. Louis, MO, USA) containing luciferin-1A2, fresh medium containing luciferin-IPA or luciferin-CEE. After 1 or 4 h of incubation, 50 μl of buffer or culture medium from each well were passed to a 96-well opaque white plate by mixing with an equal volume of the luciferin detection reagent to initiate a luminescent reaction. After 20 min of shaking at room temperature, luminescence was measured using a microplate reader (DTX880; Beckman Coulter, Inc.).