Micron and Microscopica Acta, V 01. 22, No. 3. pp. 251—252, 199!. (1739-6261)19! $3.00 +1)00 Printed in Great Britain Pergamon Press plc ELECTRON MICROSCOPICAL CHARACTERIZATION OF FRESHLY ISOLATED LIVER CELLS, AND THEIR USE FOR NORTHERN HYBRIDIZATION ANALYSIS OF CONNEC- TIVE TISSUE PROTEIN TRANSCRIPTS Albert GEERTS 1, Patricia GREENWEL3, Mike CUNNINGHAM3, Pieter DE BLESER1, Vera ROGIERS2, Eddie WISSE1 and Marcos ROJKIND3 1Laboratory for Cell Biology en Histology, and 2Laboratory for Toxicology, Free University Brussels (V.U.B.), Belgium, and 3Liver Research Center, Albert Einstein College of Medicine, Bronx, New York, USA. The literature contains conflicting data as to which cell type is the principal source of connective tissue in normal liver. In a previous study, we have examined by Northern hybridization the expression of col- lagen c.tl(I), collagen al(III), collagen al(IV), fibronectin and laminin Bi genes in freshly isolated and purified parenchymal, endothelial, Kupifer and fat-storing cells of normal rat liver [1]. We have found that parenchymal cells contain fibronectin, collagen al(III) and, to a much lower level, also collagen Cd (I) mRNAs. Fat-storing cells contain collagen ctl (III), collagen czl(IV) and laminin B! chain mRNAs. Endothelial cells contain collagen al(IV) mRNA. In the present study, we have examined by transmission electron microscopy whether freshly isolated parenchymal, endothelial, Kupifer and fat- storing cells were of sufficient purity to allow unequivocal interpretation of the obtained hybridization data. Adult male Wistar rats, weighing 300 - 400 grams, were used. Parenchymal cells were isolated and purified according to Rogiers et al [2]. Kupffer and endothelial cells were isolated and purified using centrifugal elutriation according to Van Bossuyt et al [3]. Fat-storing cells were isolated and purified by centrifugation through 13 and 11 % (w/v) Nicodenz according to Dc Bleser et a! [4]. Isolated and purified cells were characterized morphologically by transmission electron microscopy. For each cell type, preparations obtained from three different animals were examined. For transmission electron microscopy, 5 x 106 cells were resuspended in 0.5 ml of 1.5 % glutaraldehyde, brought into a small eppendorf tube and centrifuged for 4 mm at 9000 x g. The pellet was further fixed in 1 % 0s0 4, dehy- drated and embedded in Epon. 60 nm sections were cut with a diamond knife, stained with uranyl ace- tate and lead citrate and viewed using a Philips TEM 400 at 80 kV. Each cell pel’et was cut along its longitudinal axis. The cells present in each section were identified based on ultrastructural criteria [5,61. For each cell preparation, at least 500 cells were counted in randomly selected fields. Parenchymal cells were characterized by their large diameter, centrally located nucleus with large nucleolus, numerous mitochondria, glycogen rosettes and extended rough and smooth endoplasmic reti- culum (Fig. la). The most numerous contaminating cells were endothelial cells that were still attached to the parenchymal cells. Freshly isolated endothelial cells contained a bean shaped nucleus, many retracted fenestrae, and occasional lysosomes (Fig. ib). Kupifer cells were characterized by their irreg- ular nucleus, ruffled cell surface, and the presence of numerous lysosomes (Fig. ic). Fat-storing cells were characterized by the presence of cytoplasmic fat-droplets, dilated rough endoplasmic reticulum and a highly irregular cell surface (Fig. ld). Table 1. summarizes the average cellular composition of each of the cell isolates. Parenchymal, endothelial and Kupifer cells were more than 85 % pure, fat-storing cells 69.1 %. The level of contamination of parenchymal, endothelial and Kupifer cells by fat-storing cells never exceeded 2.9 %. 251