METHODS IN LABORATORY INVESTIGATION - 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 sinusoid al endothelial cells (LEC). With regard to the purity, morphology, and responsiveness of the LEC, a detailed electron microscopy study was p erformed. In addition, we developed a method of automatic detection an d analysis of the endothelial fenestrations using digitized scanning e lectron microscope images, allowing us to collect large sets of data. EXPERIMENTAL DESIGN: LEC were isolated by collagenase perfusion of the liver, isopycnic sedimentation in a two-step Percoll gradient, and se lective adherence. The purification and cultivation of LEC was evaluat ed by light and electron microscopy. The addition of ethanol to LEC cu ltures showed responsiveness of LEC. RESULTS: Purity and viability of LEC after selective adherence was 73.7 +/- 5.8% and greater than or eq ual to 95%, respectively. LEC purity was further enhanced during adher ence and spreading on collagen (type I, III). After 8 hours of culture , LEC monolayers were contaminated with less than 5% of other cells. A fter treatment with ethanol for 90 minutes, the diameters of LEC fenes trae increased approximately 10%. CONCLUSIONS: LEC isolated by this me thod provide a vital and responsive cell population enabling the study of structure and function of these cells in vitro. This method, in co mbination 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.