A MORPHOLOGICAL-STUDY OF DIFFERENTIATED HEPATOCYTES IN-VITRO

A problem traditionally encountered with primary hepatocyte cultures is their rapid dedifferentiation, which is reflected not only in decreased liver-specific functions, but also in dedifferentiated morphology: the cells flatten, depolarize, and lose many of the surface characteristics of normal hepatocytes in vivo. However, culture conditions that maintain primary rat hepatocytes in a healthy and highly differentiated state were recently developed: the hepatocytes are cultured in Chee's Medium supplemented with dexamethasone and dimethyl sulfoxide (DMSO) on collagen-coated Permanox dishes. In addition to retaining labile hepatocyte specific functions (e.g., P450 activity and albumin synthesis), these hepatocytes also have a differentiated morphology. They have numerous microvilli and are cuboidal and cluster into cords reminiscent of hepatic trabeculae, Their subcellular organelles have normal morphology, and specialized junctions and bile canaliculi form within the membranes of adjacent cells, Actin fibers cluster art these canalicular surfaces. These hepatocytes also synthesize blood clotting factors, which aggregate into fibrin meshworks between cells, Taken together, these morphological data suggest that these hepatocytes are polarized and generally have an appearance very similar to parenchymal cells in the liver, and that the same culture conditions that promote retention of liver-specific functions are also critical to the maintenance of physiological morphology, In contrast to other hepatocyte cultures, this differentiated morphology, including the polarized nature of the cells, is established without the use of serum or flexible or complex extracellular matrices and shows a close link between cellular architecture and tissue-specific function.