Bile acid uptake assay and transcellular transport assay

The transcellular transport of bile acids was measured by the translocation of fluorescently labeled bile acid (CGamF; a kind gift from Dr Hofmann) between the culture chambers as previously described in the two-chamber Transwell system (Hofmann et al., 2010; Mita et al., 2005, 2006). Briefly, after hepatic differentiation of HE-iPSCs in the two-chamber culture, hepatocyte-like cells on the Transwell membrane were pre-incubated with a transport buffer (118 mM NaCl, 23.8 mM NaHCO₃, 4.83 mM KCl, 0.96 mM KH₂PO₄, 1.20 mM MgSO₄, 12.5 mM HEPES, 5 mM glucose, 1.53 mM CaCl₂, adjusted to pH 7.4) for 20 min. Transepithelial electrical resistance (TEER) was measured with an EVOM2 probe (WPI). For the uptake assay, the cells were incubated with 10 µM CGamF for 60 min and supernatants were washed three times with PBS. Then cells were lysed with NaOH and the fluorescence intensity of the cell lysates measured at 490 nm. Fluorescence intensity was normalized to the protein amount in the cells, as measured by Bradford assay. For the basolateral to apical transport assay, 10 µM CGamF was added to the lower chamber and the transport buffer only to the upper chamber. Sixty minutes later, the transport buffer in the upper chamber was collected and fluorescence intensity measured at 490 nm. The fluorescent intensity was normalized to protein amounts of the hepatocyte-like cells. For apical-basolateral transport, CGamF was added to the upper chamber and fluorescence measured in the lower buffer.