Cryopreserved human primary hepatocytes

Cryopreserved human primary hepatocytes (Lonza) were plated overnight on collagen-coated 12-well plates at 1 × 10⁵ cells per well in MM (Lonza). 24 h after plating, cells were serum-starved in DMEM base medium (Sigma) supplemented with 1 g/L glucose (Sigma), 3.7 g/L sodium bicarbonate (Sigma), and 4 mM L-glutamine (Corning) overnight, followed by 24 h incubation in 0.3 ml glucose-production medium: DMEM base with 2 mM glutamine, 3.7 g/L sodium bicarbonate, 15 mM HEPES (ThermoFisher), 20 mM lactate (Sigma), 2 mM pyruvate (Fisher) and 0.1 mM pCPT-cAMP (Sigma). After 24 h, 50 μL of medium was removed for glucose detection with Invitrogen glucose Colorimetric Detection kit (#EIAGLUC), according to manufacturer’s protocol, and read on a plate reader (Multiskan GO, Thermo-Scientific). Because hepatocytes were extensively washed prior to cell incubation in glucose-free media for this assay, the only potential source of glucose in the media is hepatic production. The prolonged culture of cells in low glucose media prior to the assay depletes hepatocytes of glycogen stores. The media used during this assay contains high concentrations of gluconeogenic substrates, primarily lactate, favoring gluconeogenesis^{67 (link),68 (link)}.

The demographic information for the six liver tissues (three males and three female, age 44 to 73) is provided in Supplemental Table S1. Liver tissues from the six donors were pooled and used for ChIP-Seq. Cryopreserved human primary hepatocytes from two donors (both European American, one male and one female, age 32 and 62, respectively) were obtained from Lonza (Basel, Switzerland).

Cryopreserved human primary hepatocytes (Lonza) were plated overnight on collagen-coated 96-well plates at 2 × 10⁴ cells per well in MM (Lonza) and collected after 24 and 48 h of siRNA transfection. Total bile acids were measured following the protocol supplied in the Total Bile Acid Assay Kit available from Cell Biolabs (San Diego, Ca). Absorbance data was collected using the SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA) microtiter plate reader. The total bile acids were calculated by extrapolating test values to a calibration curve as described in the assay kit. The levels of Glycochenodeoxycholate were measured by mass spectroscopy, as described elsewhere^{66 (link)}. Briefly, 100 μL of acetonitrile was added to 50 μL of cell culture. The samples were vigorously vortexed and then centrifuged (22,000 × g, 2.5 min). The supernatant fraction was diluted 1:10 in 20% acetonitrile in H₂O for analysis by LC–MS/MS. Glycochenodeoxycholate (Sigma) was used to prepare a standard curve (1 nM–10,000 nM). The concentration of Glycochenodeoxycholate in the media was determined by linear regression analysis.