3-D COCULTURE OF HEPATIC SINUSOIDAL CELLS WITH PRIMARY HEPATOCYTES - DESIGN OF AN ORGANOTYPICAL MODEL

Models for cocultures of parenchymal (PC) and nonparenchymal cells (NP C) of the liver relied on mixing the cells in a two-dimensional config uration or on establishing spheroidal aggregates. In vivo hepatic nonp arenchymal cells, such as endothelial cells and Kupffer cells, are sep arated from parenchymal cells by extracellular matrix (ECM). Due to th eir location outside of the space of Disse they can form a barrier tow ard the sinusoid. Hepatocytes are attached to ECM of the space of Diss e via two opposing sinusoidal surfaces. No three-dimensional coculture model reflecting this specific microenvironment of the liver cell pla tes in vivo has been available to date. We designed a three-dimensiona l model by positioning NPC on top of PC enclosed as a monolayer within a collagen sandwich. A gas-permeable membrane support can be used to allow the supply of oxygen to the resulting cell plate also from under neath the cell layers. Morphological analysis was performed by inverse and cross-sectional studies by light microscopy, scanning, and transm ission electron microscopy of the coculture model. Cuboidal hepatocyte s formed confluent layers below the NPC layer, They regularly expresse d bile canaliculi at intercellular contact zones. Both sinusoidal surf aces expressed microprojections. Characteristic NPC including endothel ial cells, Kupffer cells, and Ito cells completely covered the second matrix layer within a week. Kupffer cells were located on top of endot helial cells. Ito cells were intermingled and could be identified by t heir intracytoplasmic lipid droplets. LPS stimulation of cocultures re sulted in a depression of albumin secretion. Phase I and phase II meta bolites of the cytochrome P-450 1A1 substrate ethoxyresorufin were gen erated independently from the presence of cocultured NPC, This study d escribes the development of a novel three-dimensional coculture model, which intends to mimic more closely the microenvironment of the hepat ic sinusoid by respecting the specific plate structure of the liver pa renchyma. The model could serve as a complex tool to study potential c ollaborations between PC and NPC of the liver. (C) 1996 Academic Press , Inc.