Abstract
The formation of multilayer aggregations of attached hepatocytes on PVLA (poly-N-p-vinylbenzyl-D-lactonamide) remarkably depended on the concentrations of supplemented EGF and insulin. Furthermore, the aggregation was synergistically accelerated by the addition of both hormones as well as calcium ion into the culture medium. Hepatocytes in the aggregation exhibited better maintenance of specific hepatocyte-functions such as the secretion of albumin and bile acid, and retained mitochondrial enzyme activity than those in the monolayer culture on collagen and fibronectin. On the other hand, DNA synthesis of hepatocytes cultured on PVLA was clearly suppressed. The formed multilayer aggregation was histologically confirmed to hold thee-dimensional structure mediated by an extracellular matrix. The formation of multilayer aggregation was suppressed by the addition of cis-OH-proline which is specific inhibitor of collagen synthesis and GRGDSP oligopeptide which competes with the specific binding site of adhesive proteins such as fibronectin and vitronectin. These results suggested that cultured hepatocytes on PVLA produced some extracellular matrices with the addition of above-mentioned hormones and then made use of them for self-assembling of the multilayer aggregation.
