4.1. Cell Lines and Culture Conditions

HCC HepG2 cells were purchased from the American Type Cultural Collection (ATCC) (Manassas, VA, USA) and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen Canada Inc., Burlington, ON, Canada). The cells were supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories Inc., Etobicoke, ON, Canada) and 1 mL gentamicin (50 µg/mL) (Gibco^TM, ThermoFisher Scientific, Missisauga, ON, Canada). CCA HuCCT1 cells were obtained from the cell culture bank of the Japan Health Sciences Foundation. They were grown in Roswell Park Memorial Institute medium (RPMI-1680, Invitrogen Canada Inc., Burlington, ON, Canada) and supplemented with 10% FBS and 1 mL gentamicin (50 µg/mL). Immortalized normal human liver THLE-3 cells were purchased from the American Type Cultural Collection (ATCC) (Manassas, VA, USA) and cultured in previously coated flasks. The cells were supplemented with 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type 1, and 0.01 mg/mL bovine serum albumin dissolved in bronchial epithelial cell growth medium (BEGM) for 24 h. The cells were cultivated in BEGM supplemented with a BEGM SingleQuot kit except for GA-1000 and epinephrine (Lonza Group Ltd., Basel, Switzerland), 10% FBS, and 1 mL gentamicin (50 µg/mL). The human embryonic kidney HEK293T cells were a gift from Dr. Judith Hugh, University of Alberta, Canada, and were maintained in Dulbecco’s modified eagle’s medium (DMEM, Invitrogen Canada Inc., Burlington, ON, Canada) supplemented with 10% FBS and 5 mL penicillin/streptomycin (100 U/mL) (PAA Laboratories). All cells were grown in a monolayer culture in their proper media and maintained in a humidified atmosphere in a 5% CO₂ incubator at 37 °C. They were passaged when they reached 70–90% confluency using trypsin plus 0.25% EDTA for 5 min in a CO₂ incubator. The Ethics Committee of the University of Alberta approved this study (Pro00020718, study title: Perturbance of Mitochondrial Function and Induction of Apoptosis in Liver Tumor Cell Lines by Seed Extract Fractions of Nigella sativa).