Hepatic differentiation of rat liver epithelial cells

Rat liver epithelial cells are cultivated at 100% confluency on 100 µg/mL rat tail collagen type I coated culture dishes in base medium and sequentially exposed to hepatogenic growth factors and cytokines. Base medium consisted of William’s E medium without glutamine supplemented with 7.33 IE/mL benzyl penicillin, 50 µg/mL streptomycin sulphate, 1 mg/mL linoleic-acid bovine serum albumin, 0.1 mM L-ascorbic acid, 0.03 mM nicotinamide, 0.25 mM sodium pyruvate and 1.623 mM L-glutamine. The hepatic differentiation procedure is as follows: days 0–2: base medium + 2% (v/v) FBS + 20 ng/mL HGF; days 3–5: base medium + 30 ng/mL HGF + 0.5% (v/v) ITS; day 6–8: base medium + 30 ng/mL HGF + 0.25 % ITS + 20 µg/L dex; days 9–11: base medium + 20 ng/mL HGF + 20 µg/L dex; days 12–14: base medium + 10 ng/mL HGF + 20 µg/L dex + 10 ng/mL OSM and from day 15 onwards: base medium + 20 µg/L dex + 10 ng/mL OSM. Cell cultures are incubated at 33 °C in a 5 % CO2 humidified atmosphere. Media were completely changed every three days, unless otherwise defined.