Large-Scale Isolation of Sinusoidal Endothelial Cells from Pig and Human Liver

Abstract Objective. Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial 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. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel–Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. Results. In 23 pigs, an average of 9 × 10 4 SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a “sandwich” configuration. Standard cultures showed an average of 90% SECs in primary cultures and 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 easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.