Assessment of a method of isolation, purification, and cultivation of rat liver sinusoidal endothelial cells.
BACKGROUND
This paper describes a simple, low cost, and rapid method for the isolation, purification, and cultivation of rat liver sinusoidal endothelial cells (LEC). With regard to the purity, morphology, and responsiveness of the LEC, a detailed electron microscopy study was performed. In addition, we developed a method of automatic detection and analysis of the endothelial fenestrations using digitized scanning electron microscope images, allowing us to collect large sets of data.


METHODS
LEC were isolated by collagenase perfusion of the liver, isopycnic sedimentation in a two-step Percoll gradient, and selective adherence. The purification and cultivation of LEC was evaluated by light and electron microscopy. The addition of ethanol to LEC cultures showed responsiveness of LEC.


RESULTS
Purity and viability of LEC after selective adherence was 73.7 +/- 5.8% and > or = 95%, respectively. LEC purity was further enhanced during adherence and spreading on collagen (type I, III). After 8 hours of culture, LEC monolayers were contaminated with less than 5% of other cells. After treatment with ethanol for 90 minutes, the diameters of LEC fenestrae increased approximately 10%.


CONCLUSIONS
LEC isolated by this method provide a vital and responsive cell population enabling the study of structure and function of these cells in vitro. This method, in combination with a computer-assisted, on-line image analysis allows the acquisition of large numbers of measurements on these cells with high accuracy and with a minimum of bias.
Laboratory for Cell Biology and Histology (VUB), Brussels-Jette, Belgium.
F
F
Braet
R
R
De Zanger
T
T
Sasaoki
M
M
Baekeland
P
P
Janssens
B
B
Smedsrød
E
E
Wisse
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D017364:Collagenases; D046508:Culture Techniques / Q000379:methods*; D004727:Endothelium / Q000166:cytology / Q000648:ultrastructure; D008099:Liver / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar
D017364:Collagenases