Morphologic and functional studies of mouse hepatocytes in primary culture

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Abstract
Mouse liver cells in primary culture were evaluated by highresolution 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 eight 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.

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