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In an attempt to demonstrate the three-dimensional cytoskeletal system of human hepatocytes, we used two kinds of tissue processing. In one, specimens were immediately fixed with 1% glutaraldehyde, saponin 0.5 mg/ml, and tannic acid 2 mg/ml (procedure A) and in the other, cytoplasmic matrix and organelles were washed out with a solution containing saponin 0.5 mg/ml and 0.15% Triton X-100, followed by fixation with glutaraldehyde (procedure B). The specimens were embedded in polyethylene glycol, sectioned at 500 nm, critical point-dried and observed in a TEM. With procedure A, a fine lattice was shown to spread throughout the cytoplasm so as to connect plasma membranes with cytoplasmic organelles, condensed around the bile canaliculi. With procedure B, microfilaments, microtubules and intermediate filaments could be clearly visualized three-dimensionally instead of the lattice structure. At a higher magnification, microvilli of canaliculus were found to contain a core microfilament which were enmeshed in a pericanalicular filament plexus. This plexus looking like the terminal web of intestinal epithelia, encircled the canaliculus and was attached to the zonula adherens. Outside the plexus, intermediate filament network was shown attached to the desmosome. These morphological findings indicated that the cytoskeleton of human hepatocytes is composed of microfilaments, microtubules, intermediate filaments and the fine lattice structure, suggesting that the bile canaliculi could contract in vivo.

Three-dimensional structure of cytoskeletal system in human hepatocytes viewed by polyethylene-glycol-embedding method.