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 therap eutic applications as the biological component of extracorporeal liver assi st devices to overcome the shortage of human hepatocytes. However, their fi nite life span and typically rapid loss of functions limit their utility. A n immortalized, nontumorigenic, highly differentiated porcine hepatocyte ce ll line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control o f the SV40 early promoter. From the established 69 clones, 23 clones displa ying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through mo re than 60 passages and 240 divisions. Albumin protein, present in early pa ssages, was lost at later passages, but albumin transcript still was detect able 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 immu nofluorescent staining and Western blot in HepLiu cells. Furthermore, maint enance of P450 functions in HepLiu cells was evidenced by diazepam and 7-et hoxycoumarin metabolites measured by HPLC. Phase II conjugative function wa s measured as acetaminophen glucuronidation. P450 dealkylase was demonstrat ed microscopically by the conversion of a nonfluorescent substrate to a flu orescent product. Both Northern blot analysis and immunofluorescent stainin g showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined imm unodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, e ven in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, live r biologic regulation studies, and artificial liver support systems.