Hepatocytes entrapped in alginate gel beads and cultured in bioreactor: Rapid repolarization and reconstitution of adhesion areas

The maintenance of the differentiated hepatocyte phenotype and its specific physiological properties is known to depend on several factors, such as ch emical signals, cell-cell and extracellular matrix molecular interactions, as well as the use of three-dimensional matrices. The entrapment of hepatoc ytes within Ca-alginate at high cell density and the culture under continuo us flow favour the development of three-dimensional organization and promot e expression of the differentiated hepatic phenotype. This system could rep resent an improvement in hepatocyte cultivation for basic studies of liver physiology and metabolism; it could also be applicable in toxicology, hepat ocyte transplantation or development of bioartificial organs. This report d escribes the effect of alginate entrapment and culture in a bioreactor on h epatocyte aggregate formation, with particular attention to the reestablish ment of cell polarity, cell junctions and three-dimensional re-organization of the cytoskeleton. Oxygen supply and cell oxygen consumption rate were m onitored in order to evaluate possible changes in hepatocyte energy require ment. Our data show that after only 6 h of perfusion in the bioreactor, act in and cytokeratin localize along the adhesion areas of the plasma membrane , in which reconstituted bile canaliculi were also observed. Moreover, the presence of connexin at the level of joined membranes of neighbouring cells suggests the establishment of gap junctions between hepatocytes. After the first 30 min of perfusion the oxygen consumption rate remained constant th roughout the experimental period. Copyright (C) 2001 S. Karger AG, Basel.