Isolation and culture of hepatocytes from human liver of immediate autopsy

Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intact lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 X 10(6) cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochrome p450 and 536 pmol/mg protein cytochrome b5 were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture.