Morphologic and functional studies of mouse hepatocytes in primary culture

Mouse liver cells in primary culture were evaluated by high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). Cells after 2 hours of culture in L-15 medium supplemented with 10% fetal bovine serum were spherical in shape, and were either individual or in small clusters of up to ten cells. Following 1 day in culture, hepatocytes were flattened and usually found in groups. Bile canaliculus-like structures were apparent between hepatocytes. Tight junctions and desmosomes were also present along adjacent plasma membranes. Autophagic vacuoles were seen within the cytoplasm. After 2 days in culture, hepatocytes appeared more elongated and flattened than in earlier sampling periods. Both autophagic and clear vacuoles were seen in the cytoplasm. Mitochondria were present in a variety of shapes and sizes. Small bundles of microfilaments were frequently seen in the basal region of cross-sectioned cells. From the fourth until the eighth day in culture, hepatocytes displayed further progression of the morphologic changes seen after 2 days. Nuclear elongation and the projection of cytoplasmic pseudoinclusions into the nucleus were also evident after 4 days. Cytoplasmic and nuclear changes were eventually observed in all hepatocytes by the eighth day of culture. DNA synthesis in the cells during culture was investigated by autoradiography. The percentage of S-phase labeled cells was 0.1% after 1 day of culture. The labeling index increased to 1.02%, 3.14%, and 5.88% after 2, 4, and 6 days of culture, respectively. Synthesis of albumin by the liver cells was also detectable during the first 8 days of primary culture. A gradual drop in albumin synthesis was noted with increased time in culture. The percentage of hepatocytes that histochemically stained for gamma glutamyl transpeptidase (GGT) progressively increased from 0.01% of the cells after 2 hours culture to 3.14% of the cells after 8 days of culture.