Reconstruction of 3D stacked-up structures by rat small hepatocytes on microporous membranes

The three-dimensional (3D) culture of hepatocytes is essential for the reconstruction of functional hepatic tissues in vitro. In the present experiment, we developed a 3D-culture method in order to reconstruct hepatic cordlike structures by stacking up two-dimensional (2D) tissues composed of rat small hepatocytes (SHs), which are hepatic progenitor cells. Pairs of membranes were prepared and the cells were separately cultured on each membrane. After the SH colonies had developed, one membrane was inverted on top of the other to form an SH bilayer. Thereafter, we investigated whether the stacked cells were organized into differentiated tissues. In the 3D stacked-up structures, bile canaliculi (BC) started to form and gradually developed into anastomosing networks. Transmission electron microscopy revealed that the SHs of the upper and lower layers adhered to one another, and that BC formed between them. Bile canalicular proteins localized on the lumina of the tubular structures. Furthermore, the cells within the structures exhibited mRNA transcription of the hepatic-differentiation markers and maintained a relatively high level of albumin secretion. We conclude that highly differentiated 3D tissues, including functional BC, can be reconstructed by stacking up layers of SHs. This 3D stacked-up culture is useful for the reconstruction of tissue-engineered livers.