Rifampicin solution

The Cytochrome P450 enzymes activity was performed using the P450-GloTM Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions. We tested the activity of different P450 enzymes, in particular the CYP2B6 (P450-Glo CYP2B6 – V8321/2 – Promega, Madison, WI), CYP3A4 (P450-Glo CYP3A4 (Luciferin-IPA) – V9001/2 – Promega, Madison, WI), and the CYP1A2 (P450-Glo CYP1A2 Induction/Inhibition – V8421/2 – Promega, Madison, WI) by incubating them with different inducers. For the CYP2B6 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 1000 μM Phenobarbital solution (Sigma), or DMSO (0.1%) for 48 hours. For the CYP3A4 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 20 μM Rifampicin solution (Sigma), or DMSO (0.1%) for 48 hours. For the CYP1A2 activity assay, undifferentiated hiPSC, primary hepatocytes and differentiated HLCs were incubated with basal medium containing 50 μM Omeprazole solution (Sigma), or DMSO (0.1%) for 48 hours. Measurement of the activity of each enzyme was performed by reading the luminescence using a luminometer (Synergy H1 Hybrid Reader - Biotek) according to the manufacturer’s instructions. All the experiments were performed in triplicate.

Following the manufacturer's protocol, cytochrome P450 enzyme activity was measured using a P450-Glo Assay Kit (Promega, Madison, WI, USA). Approximately 100 aggregates were isolated from each group of culture systems at the end of the hepatic differentiation. For the CYP3A4 assay, the aggregates were incubated in an hepatocyte culture medium (HCM) with BulletKit supplementation and 20 μM rifampicin solution (Sigma-Aldrich) for 48 h. CYP1A2 activity was evaluated by incubating the aggregates in the HCM Hepatocyte Culture Medium with BulletKit containing 50 μM omeprazole solution (Sigma-Aldrich) for 48 h. For the CYP2B6 activity assay, the aggregates were incubated with the HCM with BulletKit supplement containing 1000 μM phenobarbital solution (Sigma-Aldrich, USA) for 48 h. The CYP activity value was normalized to the number of cells tested. The activity of CYP2B6 was tested using P450-Glo CYP2B6 (Promega, Madison, WI, USA), CYP3A4 using P450-Glo CYP3A4 (Promega), and CYP1A2 using P450-Glo CYP1A2 (Promega).