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 liver failure to transplant. However, a major drawback of the existing technology 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 acetaminophen metabolism. To investigate the suitability of HepLiu cells for artificial liver support, morphology, as well as xenobiotic metabolism, was studied 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 inoculation of the LAD until Day 10 when proliferation appeared to cease. Ultrastructural analysis demonstrated that HepLiu cells retained many of the features of primary hepatocytes including desmosomes that sealed bile canalicular-like structures and junctional complexes (intermediate, gap junctions) that appeared concentrated in the paracanalicular areas. Unlike primary porcine hepatocytes, HepLiu cells retained drug metabolic function throughout the 25 day culture period. Diazepam metabolism by HepLiu cells was consistently higher than that of primary cells. Acetaminophen metabolism persisted throughout the 25 day period albeit at a much lower level than the primary cells exhibited on Days 1 or 2. In conclusion, we have shown that HepLiu cells proliferate to occupy the intercapillary space of perfused hollow-fiber LADs following inoculation, and retain their metabolic capacity for Phase I and Phase II detoxification reactions in perfusion culture. Our findings suggest that HepLiu cells may provide an alternative to primary porcine hepatocytes as the cellular component of bioartificial liver support systems.
Liu
J
J
MultiCell Associates, Inc., 55 Access Road, Warwick, RI 02886, U.S.A. JinLiu@multicellassociates.com
Jauregui
H O
HO
Faris
R A
RA
Santangini
H A
HA
Trenkler
D M
DM
Silva
P G
PG
Garcia
K M
KM
D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line / Q000637:transplantation; D017690:Cell Transplantation / Q000379:methods; D004195:Disease Models, Animal; D022781:Hepatocytes / Q000378:metabolism* / Q000637:transplantation* / Q000648:ultrastructure; D017093:Liver Failure / Q000628:therapy; D019164:Liver, Artificial; D012680:Sensitivity and Specificity; D013552:Swine