Abstract
Our laboratory has been developing hepatocyte-based bioartificial liver support for over 2 decades. The current practice of primary porcine hepatocyte (PPH) procurement is costly, labor intensive and yields hepatocytes of variable detoxification capacity. These limitations led us to develop the HepLiu immortalized hepatocyte cell line, which is nontumorigenic and exhibits hepatic detoxification functions in monolayer cultures. To determine the suitability of HepLiu cells for artificial liver support, their drug metabolic activity [diazepam (Dz), acetaminophen (APAP) and 7-ethoxycoumarin (7EC)] as well as their morphology were evaluated in perfused hollow fiber devices. HepLiu cells and PPH were seeded in either Nunc Permanox 60mm plates or polysulfone hollow fibers chambers. To assess detoxification, the cells in either culture condition were incubated with the test drug (50 μg/ml Dz or 7EC; or 5mM APAP) for 3 hours and metabolites were assayed by HPLC. Samples were fixed and embedded in Epon 812. Ultrastructural studies were performed in a Philips 300 electron microscope. Results: Unlike primary hepatocytes, HepLiu cells retained drug metabolic function through day 25 in perfused chambers. HepLiu cells proliferated rapidly through day 6 of culture, when cell number reached a plateau, remaining steady through day 25. At any timepoint beyond 2 days, metabolism of APAP and Dz was higher in HepLiu cells than in PPH. Transmission electron microscopy showed HepLiu cells in LAD filled the entire intercapillary space displaying desmosomes that sealed bile canaliculi (BC). Adjacent junctional complexes (intermediate, gap junctions and desmosomes) remained concentrated in the para-canalicular areas with numerous microvilli at the cell surface interfacing the fibers and connecting with many BC. Cell organelles such as mitochondria, Golgi, SER, RER, lysosomes, glycogen and lipid vesicles. HepLiu cells proliferate freely to occupy the intercapillary space of LAD and retain phase I and II detoxification functions demonstrating the feasibility of their use in bioartificial livers.
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