Hepatocyte Isolation and Culture Protocol

Hepatocytes were isolated by in situ collagenase digestion (50 U/L collagenase NB 4G, Serva Electrophoresis, Heidelberg, Germany) and further purification steps as described earlier.^{2 (link),14 (link)} Viability after isolation was routinely determined by trypan blue exclusion to normalize viable cell number to 10⁶ cells for seeding/cold storage. Adherent control cultures were obtained as described earlier,^{14 (link)} seeding 10⁶ viable cells per well onto collagen-coated 6-well plates in supplemented Leibovitz L-15 cell culture medium. After 2 h, cell cultures were washed 3 times with warm Hanks balanced salt solution (HBSS) and supplied with 2 mL of fresh medium.