2.2. Primary Mouse Hepatocytes Isolation
The primary hepatocyte maintenance media were as follows: Williams’ Medium E with supplements (WEM), Williams’ medium E containing 1× B27, 1× Glutamax, and 1× penicillin-streptomycin; 5C condition (5C): WEM supplemented with Forskolin (20 µM), SB431542 (10 µM), IWP2 (0.5 µM), DAPT (5 µM), and LDN193189 (0.1 µM); Hepatocyte Culture Medium (HCM): manufactured by Lonza (CC3198); Proliferation human hepatocytes condition (ProliHH): Advanced DMEM/F12 supplemented with 1 mM N-acetylcysteine, 1× B27 (minus vitamin A), 1× N2 supplement, 10 nM gastrin I, 25 µg/mL recombinant Human Wnt-3a Protein, 50 ng/mL EGF, 2 ng/mL FGF10, 25 ng/mL recombinant human HGF, 25 ng/mL human recombinant bFGF, 5 µM A83-01, 10 µM Y-27632, 1% penicillin-streptomycin, and 1% fetal bovine serum.
2.6. Electroporation
Electroporation was performed using a P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza, Basel, Switzerland). According to the manufacturer’s instructions, 600,000 freshly isolated mouse hepatocytes were resuspended in 100 µL of Lonza electroporation buffer P3 (82 µL Nucleofector Solution and 18 µL Supplement) containing 5 µg of DNA. The cell suspension was then transferred into NucelocuvetteTM vessels and electroporated using a Lonza 4D-NucleofectorTM core unit with the DS-150 program. After electroporation, the cells were resuspended in plating medium and seeded onto collagen I-coated plates for at least 3 h. The plating medium was then replaced with pre-warmed HCM. After 72 h, images were captured using an Olympus IX73 fluorescence microscope and the transfection efficiency was quantified using Image J software (version 1.53e).