Growth and metabolic activity of immortalized porcine hepatocytes in extracorporeal hollow-fiber liver assist devices

The development of a cell based extracorporeal liver assist device offers a promising clinical approach to bridge individuals suffering from acute liv er failure to transplant. However, a major drawback of the existing technol ogy is the lack of a continuous supply of well differentiated hepatocytes. Although some investigators have used primary porcine cells, this approach demands costly, labor-intensive isolation procedures and yields cells with inconsistent detoxification capacity. The limitations of primary cells led us to develop the HepLiu immortalized porcine hepatocyte cell line for use in liver assist devices (LADs). HepLiu cells are nontumorigenic and exhibit multiple hepatic detoxification functions including diazepam and acetamino phen metabolism. To investigate the suitability of HepLiu cells for artific ial liver support, morphology, as well as xenobiotic metabolism, was studie d in perfused polysulfone hollow-fiber LADs. HepLiu cells were cultured in the intercapillary space of a prototype LAD, and the metabolism of diazepam , acetaminophen, and 7-ethoxycoumarin was evaluated over 25 days in culture . Our results indicated that HepLiu cells proliferated rapidly following in oculation of the LAD until Day 10 when proliferation appeared to cease. Ult rastructural analysis demonstrated that HepLiu cells retained many of the f eatures of primary hepatocytes including desmosomes that sealed bile canali cular-like structures and junctional complexes (intermediate, gap junctions ) that appeared concentrated in the paracanalicular areas. Unlike primary p orcine hepatocytes, HepLiu cells retained drug metabolic function throughou t the 25 day culture period. Diazepam metabolism by HepLiu cells was consis tently higher than that of primary cells. Acetaminophen metabolism persiste d throughout the 25 day period albeit at a much lower level than the primar y cells exhibited on Days 1 or 2. In conclusion, we have shown that HepLiu cells proliferate to occupy the intercapillary space of perfused hollow-fib er LADs following inoculation, and retain their metabolic capacity for Phas e I and Phase II detoxification reactions in perfusion culture. Our finding s suggest that HepLiu cells may provide an alternative to primary porcine h epatocytes as the cellular component of bioartificial liver support systems .