Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro

Abstract

Cell sheet stratification technology has been used for reconstituting highly functional three-dimensional (3D) hepatic tissues in vitro. Triple-layered hepatic tissues with a hepatocyte-specific polarity were fabricated by sandwiching a hepatocyte sheet (Hep sheet) between two endothelial cell (EC) sheets. The morphological and functional characteristics of the triple-layered hepatic construct (EC-Hep-EC) were evaluated and compared with those of a double-layered hepatic construct with a single EC sheet (Hep-EC) and a Hep sheet only. Transmission electron microscope (TEM) observations revealed that the extracellular matrix was observed to be deposited in the space between the ECs and hepatocytes on both the upper and lower sides of the hepatocytes in the EC-Hep-EC construct. Immunohistochemistry with basolateral (CD147) and apical [multidrug resistance-associated protein (MRP2)] membrane polarity markers clearly showed the recovery of in vivo-like hepatocyte polarization in the EC-Hep-EC group. In addition, hepatocyte-specific functions, including albumin secretion, ammonia removal and the induction of cytochrome P450, were also highly preserved. The presented technology for stratifying multiple cell sheets was simple in operation and successfully reproduced both the heterotypic/homotypic cell-cell and cell-matrix interactions with the inherent hepatocyte configurations, thus closely mimicking the in vivo environment. The triple-layered 3D hepatic constructs could therefore be valuable as a new experiment tool for drug-screening tests, an implantable tissue model for cell-based therapies and an efficient culture platform for bioartificial liver devices. Copyright © 2015 John Wiley & Sons, Ltd.

Keywords: 3D tissue; cell sheet; cell-cell interaction; co-culture system; hepatic function; hepatocyte polarity.