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


CONCLUSIONS
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.
Gerlach
J C
JC
Surgical Department, Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany. joerg.gerlach@charite.de
Zeilinger
K
K
Spatkowski
G
G
Hentschel
F
F
Schnoy
N
N
Kolbeck
S
S
Schindler
R K
RK
Neuhaus
P
P
D000818:Animals; D001672:Biocompatible Materials; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen; D017364:Collagenases; D004338:Drug Combinations; D004727:Endothelium / Q000166:cytology; D022781:Hepatocytes / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D007797:Laminin; D008099:Liver / Q000166:cytology*; D008297:Male; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D010477:Perfusion; D011509:Proteoglycans; D011971:Receptors, Immunologic / Q000032:analysis; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B; D013552:Swine
D001672:Biocompatible Materials; D004338:Drug Combinations; D007797:Laminin; D008565:Membrane Proteins; D011509:Proteoglycans; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C059485:matrigel; D003094:Collagen; D017364:Collagenases