Large-scale isolation of sinusoidal endothelial cells from pig and human liver

Objective. Hepatic in vitro studies, like those on hypoxia/reperfusion inju ry in liver transplants, demand large numbers of cultivated sinusoidal endo thelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. Methods. SECs were isolated employing a four-step collagenase perfusion. Th e sinusoidal character of the cells was validated by transmission and scann ing electron microscopy, exclusion of Weibel-Palade bodies and factor VIII- related antigen, expression of scavenger receptor, and incorporation of lat ex beads. Results. In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multi layer cocultures of isolated SECs and hepatocytes in a "sandwich" configura tion. Standard cultures showed an average of 90% SECs in primary cultures a nd 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. Conclusion. With the four-step collagenase perfusion it is possible to easi ly isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ. (C) 2001 Academic Press.