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.

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems.

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C. University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.

Flow cytometric characterization of isolated porcine hepatocyte suspensions for liver support.

The high yield hepatocyte isolation necessary for hybrid liver assist devices (LAD) unavoidably increases contamination by nonparenchymal cells and depresses hepatocyte viability and functions. We have developed a flow cytometric procedure that improves quality control of the isolations. Cells present in these preparations were labeled by immunofluorescent antibody staining against cytokeratin 8, 18 as well as vimentin to identify hepatocytes, fibroblasts, and endothelial cells. Antibody staining against albumin and carbamoylphosphate synthetase allowed assessment of levels of albumin and carbamoylphosphate synthetase based on the hepatocyte relative fluorescence intensity. Hepatocyte P450 enzyme activity was measured by its ability to convert 5,6-methoxycarbonylfluorescein, a nonfluorescent substrate, to an intracellular fluorescent product. Flow cytometric methods of cell type identification and cell function assessment are fast and accurate and can be applied to commercial cell production. They may also provide an avenue for the enrichment of otherwise heterogeneous hepatocyte suspensions with cells presenting the specific functions desired for an hybrid liver assist devices.

Isolation and culture of porcine hepatocytes for artificial liver support.

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from approximately 12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.

In vivo evaluation of a hollow fiber liver assist device.

Orthotopic liver transplantation is the only effective form of therapy currently available for patients with fulminant hepatic failure (FHF). The use of an extracorporeal (EC) liver assist device (LAD) may result in improved presurgical clinical management. Alternatively, patients treated with LADs could avoid the transplantation procedure if they are able to regenerate a critical mass of hepatocytes that will sustain functional viability. In this study, the efficacy of a prototype hollow fiber LAD seeded with rabbit hepatocytes was assessed in vivo by the use of two different animal models: (1) normal rabbits injected with diazepam or lidocaine, and (2) a galactosamine (Gal)-intoxicated rabbit model of FHF. The EC LAD clearly decreased the blood levels of the two drugs and significantly generated diazepam and lidocaine metabolites indicating the maintenance of active P450 forms in the cellular component of the devices. A 6-hour EC treatment significantly increased the survival time and delayed the onset of hepatic encephalopathy (HE) in the Gal-intoxicated rabbits. Histological evaluations of postmortem livers showed greater hepatocyte regenerative activity in the animals treated with hepatocyte-seeded LADs than in the two control groups, e.g., rabbits not treated or treated with unseeded devices. These findings support the concept that a microporous hollow fiber LAD seeded with rabbit hepatocytes is able to sustain drug detoxification in vivo as well as to modify the course of FHF in a well-characterized animal model.

Primary cultures of rat hepatocytes in hollow fiber chambers.

Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O2-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P < or = 0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O2-enriched conditions to maintain long-term P450 activity of rat hepatocytes.