Hepatocytes (University of Nebraska Medical Center)

Human hepatocytes were isolated by collagenase perfusion of donated livers. A single catheter was inserted into the hepatic vein and the liver was then perfused (30 min) with a calcium-free buffer (30 min) followed by a second buffer containing calcium, collagenase, and bovine serum albumin (0.5%). The calcium-free step helps separate intercellular junctions (which require calcium) while the cells were completely separated by the exposure to collagenase, at 37 °C. Plate cells in medium containing calf serum (10%). Switch to serum-free medium (SFM) as soon as possible (4-12 hr) and maintain in SFM. Culture dishes are usually precoated with type 1 collagen or gelatin 30 min prior to addition of the cells.