Objective: The aim of this study is to elucidate the temporal changes of the cell motility and the localization of contractile proteins in isolated hepatocytes after primary monolayer cultures.
Methods: Cultured hepatocyte couplets and triplets were observed at 4 hr and 24 hr with time-lapse video recording using video-enhanced contrast, differential interference contact (VEC-DIC) microscopy. The distribution of actin and myosin was examined by immunofluorescence, transmission electron microscopy, and electron microscopy using a whole cell mount.
Results: In 4 hr-cultured hepatocyte couplets, bile canalicular contractions, mediated by the surrounding actin and myosin, were spontaneous and forceful. At 24 hr, the cells spread and flattened out, and VECDIC microscopy revealed rope-like fibers, possibly stress fibers in the flattened cytoplasm. Stress fiber motilities were active and independent of other cell movements. The saltatory movement of the vesicles in the cytoplasm was clearly visualized with time-lapse recording. However, the bile canaliculi between two hepatocytes were closed at 24 hr, and the canalicular contractions were no longer seen. Actin and myosin were found in the same area as stress fibers were observed by VEC-DIC microscopy.
Conclusions: It was possible to visualize high-resolution images of the dynamic cell function in the living hepatocytes, using VEC-DIC microscopy with time-lapse recording. The changes in the cell motility pattern may be ascribed to the reorganization of the contractile proteins in isolated hepatocytes after monolayer cultures. Four hr-cultured hepatocyte couplets are considered to be similar to the liver in a living state in terms of the cell morphology and the physiological motility function of the canaliculi.