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Updated: Aug 4, 2026
Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells
Published on: March 30, 2016
Large-scale isolation of sinusoidal endothelial cells from pig and human liver
J C Gerlach1, K Zeilinger, G Spatkowski
1Surgical Department, Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany. joerg.gerlach@charite.de
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 x 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.
