For determination of albumin secretion, a culture medium from differentiated organoids was collected at day 14 of differentiation. Protein in the medium was concentrated using Amicon Ultra centrifugal filters (Millipore), and the amount of albumin was measured using a DxC-600 Beckman (Beckman Coulter). The values were normalized for total cell number. For measurement of cytochrome P450 activity, 14-day-differentiated organoids were removed from Matrigel and incubated in 50 μM luciferin-PFBE substrate (Promega) in hepatozyme medium (Gibco) containing 10% FBS for 8 hr at 37°C. Cyp3a activity was then measured with a luminometer using the P450-Glo cytochrome P450 assay kit according to the manufacturer’s instructions (Promega). For comparative analysis, freshly isolated canine hepatocytes were used (Arends et al., 2009 (link)). Furthermore, human cell lines HepG2 (cultured in DMEM with 10% FBS) and Huh 7 (cultured in DMEM with 10% FBS) were included for comparative functional analysis.
For measurement of the expression of hepatic enzymes in differentiated organoids, cells were lysed in milliQ at day 14 and stored at −20°C. ALT and AST were measured using the DxC-600 Beckman (Beckman Coulter) standard protocols, and values were corrected for total cell counts.
For measurement of the expression of hepatic enzymes in differentiated organoids, cells were lysed in milliQ at day 14 and stored at −20°C. ALT and AST were measured using the DxC-600 Beckman (Beckman Coulter) standard protocols, and values were corrected for total cell counts.