Formation of bile canaliculi in long‐term primary cultures of adult rat hepatocytes on permeable membrane: an ultrastructural study

Abstract

Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.

Development and maintenance of bile canaliculi in vitro and in vivo

“…There also appears to be an interplay between ECM receptor occupancy and cell shape that is essential in maintaining the differentiated polarized hepatocyte phenotype (Singhvi et al, 1994) see Figure 1. Growth of monolayer cultures on collagen-coated permeable membranes (Arterburn et al, 1995;Guery et al, 1995) or in perfused matrix capillary systems (Gerlach et al, 1995) maintain canalicular structures, indicating that nutrient evailability at the basolateral surface also may be a significant factor.…”

Structural Polarity and Functional Bile Canaliculi in Rat Hepatocyte Spheroids

“…Monolayer cultures of hepatocytes on extracellular matrix proteins adopt a flat morphology [13,14], and the development of bile canalicular structures in these cultures is rare, heterogeneous, and short-lived [12,15]. Several investigators have reported improved maintenance of archetypal polarity [13,16] and bile canaliculi formation in extracellular matrix sandwich cultures [14,15,17], on collagen-coated porous membranes [18], and in hepatoma-derived hybrid cell lines [19 -21]. However, the restraints of such two-dimensional systems hinder the development of anastomosing channels among many cells.…”