Layered Hepatocytes and Endothelial Cells on a Transwell Membrane: Toward Engineering the Liver Sinusoid

Abstract

This paper presents a novel liver model platform that mimics the liver sinusoid, a functional unit of the liver where most liver activities occur. A key component of the current liver model is a layered co-culture of primary rat hepatocytes (PRH) and primary rat liver sinusoidal endothelial cells (LSEC) or a bovine aortic endothelial cells (BAEC) as an alternative. Poly-dimethylsiloxane (PDMS) microchannels were fabricated and attached to transwell membranes that contain submicroscale pores. Cells were cultured either on one side or on both sides of the transwell membrane, and in both cases cells formed confluent layers. A thin matrigel coating or micro porous membrane was applied between the two cell layers in order to mimic the Space of Disse. We used three different methods to check cell viability: recombinant adenovirus expressing green fluorescent protein, mito-tracker red to stain live mitochondria, and an expression plasmid expressing red fluorescent protein (RFP). It was shown that PRH retained normal morphology and remained viable for about 3 days with BAEC in the PDMS microchannel, about 57 days with BAEC on the transwell, and about 39 days with primary LSEC on the transwell. Preliminary observation suggests that there is formation of structures between hepatocytes that appear similar to bile canaliculi when PRH are co-cultured with endothelial cells. The layered co-culture system seems to be a promising method to generate accurate liver models.