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

• Published in: The Journal of surgical research 2001-09, Vol.100 (1), p.39-45
• Main Authors: Gerlach, Jörg C., Zeilinger, Katrin, Spatkowski, Gabriele, Hentschel, Florian, Schnoy, Norbert, Kolbeck, Stefan, Schindler, Rudolf K., Neuhaus, Peter
• Format: Article
• Language: English
• Subjects: Animals; Biocompatible Materials; Biological and medical sciences; Cell Separation - instrumentation; Cell Separation - methods; Cells, Cultured; Coculture Techniques; Collagen; Collagenases; Drug Combinations; Endothelium - cytology; Gastroenterology. Liver. Pancreas. Abdomen; Hepatocytes - chemistry; Hepatocytes - ultrastructure; human liver; Humans; in vitro culture; isolation; Laminin; Liver - cytology; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Male; Medical sciences; Membrane Proteins; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Other diseases. Semiology; Perfusion; pig liver; Proteoglycans; Receptors, Immunologic - analysis; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; sinusoidal endothelial cells; Swine
• ISSN: 0022-4804; 1095-8673
• DOI: 10.1006/jsre.2001.6224

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 × 104 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.