Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes.

The ultrastructure of primary hepatocytes cultured for 2 to 17 days on floating collagen membrane was evaluated. A pellet of the hepatic cell suspension used to inoculate the collagen membranes contained some single cells and many aggregates of two, three, or four cells. Desmosomes were split during the perfusion, but tight junctions and gap junctions remained intact. By 2 days in culture, the hepatocytes had formed a monolayer of cells of polygonal shape with newly synthesized desmosomes between cells. Since the flexible floating collagen membrane decreases in size as the monolayer forms, the hepatocytes do not flatten out, as is characteristic of cells cultured on a rigid substrate. Hepatocytes in culture for 10 days or less exhibited large lamellar arrays of rough endoplasmic reticulum, well developed Golgi complexes, and structures resembling bile canaliculi, which possess tight junctions and desmosomes separating them from the intercellular space. Microfilaments oriented parallel to the plasma membranes of adjoining cells and in an intermeshed network at the edge of the monolayer and beneath the plasma membrane bordering the medium increased in size and number in older cultures. After 17 days in culture, the cells maintained tight junctions, desmosomes, Golgi complexes, and rough endoplasmic reticulum in small lamellar stacks and in small vesicles. Since hepatocytes on the floating collagen membrane retain most of the subcellular structural elements characteristic of normally functioning hepatocytes for 2.5 weeks, this system may be valuable for future experiments involving drug metabolism and carcinogenesis in vitro.