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stablishment and Characterization of Immortalized Porcineepatocytes for the Study of Hepatocyte Xenotransplantation
. Pan, W. Du, X. Yu, G. Sheng, H. Cao, C. Yu, G. Lv, H. Huang, Y. Chen, J. Li, and L.J. Li
ABSTRACT
Background. In light of the critical shortage of donor livers, xenogeneic sources offer thebest alternative to human hepatocytes for the treatment of acute liver failure. This studyinvestigated whether a combination of simian virus 40 large T antigen (SV40 LT) andhuman telomerase catalytic subunit (hTERT) genes could immortalize primary porcinehepatocytes that could reverse acute liver failure (ALF) in rats.Methods. We cotransfected SV40 LT and hTERT genes into primary porcine hepato-cytes to examine the features of the transfected cell lines. We characterized the potentiallytherapeutic effect of immortalized porcine hepatocytes in a rat model of ALF induced by90% hepatectomy.Results. An immortalized porcine hepatocyte cell line, HepLi, was expanded by �250passages. HepLi cells maintained the defining characteristics of primary porcine hepato-cytes, including porcine albumin secretion, urea production, and diazepam metabolism.Intrasplenic transplantation of HepLi cells significantly improved liver function, andsignificantly prolonging the survival of rats with ALF.Conclusions. Cotransfection of SV40 LT and hTERT immortalized primary porcinehepatocytes without tumorigenicity in vitro. The Immortalized porcine hepatocytes served
as a potential cell resource for xenotransplantation.pTlpeorfe
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lthough orthotopic liver transplantation representsthe ultimate therapy for patients with acute or
erminal liver failure; it remains a highly costly and,omplex procedure that is, hampered by the scarcity ofonor livers.1,2 Given the potential of the liver to regen-rate, research has demonstrated that hepatocyte trans-lantation (HT) can potentially serve to treat liverailure3,4 as a bridge to or an avoidance of transplanta-ion altogether. However, the development of clinical HTemains limited by the scarcity of available donor liversnd the shortcoming that primary human hepatocytesannot be readily expanded in vitro. To overcome theseroblems, some researchers have successfully xenotrans-lanted porcine hepatocytes.5,6
Primary porcine hepatocytes serve as a xenogeneic sourcer as the biologic component of extracorporeal liver assistevices.7,8 However, primary porcine hepatocyte culturesre difficult to grow and have a short lifespan in vitro, losinghenotypic expression and total cytochrome P450 proteins
n culture.9–11 Therefore, a significant concern is how to
xtend their replicative potential. z2010 by Elsevier Inc. All rights reserved.60 Park Avenue South, New York, NY 10010-1710
ransplantation Proceedings, 42, 1899–1906 (2010)
To become immortal primary cells must overcome tworoliferative blockades in vitro: senescence and crisis.ransfection with viral oncogenes, such as simian virus 40
arge T antigen (SV40 LT), enables an extended lifespan ofrimary cells beyond senescence. The transfected cellsventually succumb to crisis. However, ectopic expressionf the human telomerase catalytic subunit (hTERT) canescue postsenescent cells to acquire replicative immortalityrom crisis. Moreover, studies have demonstrated thatctopic expression of hTERT in combination with SV40 LT
From the First Affiliated Hospital, College of Medicine, Stateey Laboratory for Diagnosis and Treatment of Infectious Dis-ases, Zhejiang University, Zhejiang, China.Supported by grants from the National High Tech Research
nd Development (863) Program of China (no. H40803), theational Natural Science Foundation of China (no. 30630023),nd the Zhejiang Health Science Foundation (no. 2007A081).Address reprint requests to Professor Lan Juan Li, State Key
aboratory for Diagnosis and Treatment of Infectious Diseases,ingchun Road 79, Hangzhou, Zhejiang, China. E-mail: ljli@
ju.edu.cn
0041-1345/–see front matterdoi:10.1016/j.transproceed.2009.11.043
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fficiently generates immortal cell lines.12–14 However, itas unknown whether the combination of SV40LT andTERT genes would result in immortalization of freshly
solated primary porcine hepatocytes.In the present study, we sought to establish immortalized
orcine hepatoctyes that could be expanded in culture, bysing retrovirus-mediated gene transfer of SV40LT andTERT. Immortalized porcine hepatocytes cell lines grewontinuously, maintaining the characteristics of primaryorcine hepatocytes. Furthermore, intrasplenic transplan-ation of immortalized porcine hepatocytes saved rats fromLF induced by 90% hepatectomy.
ATERIALS AND METHODSonstruct Retroviral Vectors Containing hTERTr SV40 LT and Producing Retrovirus
T67 retroviral packing cells (Clontech, Palo Alto, Calif, USA)ultured in Dulbecco Modified Eagle Medium (DMEM; Gibco,rand Island, USA) with 10% fetal calf serum (Gibco) were
ransfected with the recombinant retroviral constructs (pLXSN–V40 LT and pLPCX-hTERT; pLPCX and pLXSN retroviralectors; Clontech) by using the calcium phosphate (Invitrogen,arlsbad, Calif, USA) method. We screened the cells with 500g/mL G418 (Clontech) and 4 �g/mL puromycin (Clontech) at 72ours after transfection. We selected and subcultured both neomy-in- resistant cells and puromycin-resistant cells. Viral titers of upo 2.0 � 107 and, 8.0 � 105 colony-forming units per milliliter werestimated by analysis of NIH 3T3 cells, respectively. The superna-ants containing the recombinant retrovirus were harvested, fil-ered through a 0.45 �m filter, and frozen at �80°C until use as thenfectious source for immortalization.
solation of Primary Porcine Hepatocytes
ur Animal Care Ethics Committee approved the experimentalrotocols, which were within the guidelines for humane care of
aboratory animals. Porcine hepatocytes isolated from adult maleigs were purchased from the Academy of Agriculture Science ofhejiang Province, using a modified 4-step collagenase perfusionethod as described elsewhere.15,16 The freshly isolated hepato-
ytes were cultured in plastic flasks (Corning, USA) at a density of.0 � 104 cells/mL with DMEM supplemented with 10% calferum, 2 mmol/L L-glutamine, 15 mmol/L HEPES, 1.5 mg/Lnsulin, 10,000 U/L penicillin G, and 100 mg/L streptomycin (pH.6). Incubation was performed at 37°C in humidified conditions of5% air and 5% CO2.
mmortalization of Porcine Hepatocytes
fter a 4-hour attachment phase, the culture was incubated at 37°Cn a 95% air/5% CO2 atmosphere overnight with a change there-fter to fresh medium. Hepatocytes were infected with a 100 �Liral stock per plate in the presence of 8 �g/mL polybrene (Aldrichhemical, USA) at 37°C for 24 hours. After the virus-containingedium was aspirated, the cultures were maintained in DMEM
upplemented with 10% calf serum for 72 hours before selectingeomycin-resistant hepatocytes by adding the neomycin analogue418 (Sigma) at 400 �g/mL. G418-resistant colonies isolated using
loning cylinders were subcultured by trypsinization. When culturesecame 70% confluent, the cells were again infected with 100 �L of
nother viral stock per plate in the presence of 8 �g/mL polybrene aAldrich Chemical) at 37°C for 24 hours. Puromycin-resistantepatocytes were selected, isolated, and expanded by subcultureor further studies.
lectron Microscopy
he ultrastructure of immortalized porcine hepatocyte cells wasbserved by scanning (SEM) and transmission (TEM) electronicroscopy. We used standard techniques for specimen prepara-
ion for SEM. Cells cultured on collagen-coated glass coverslipsere fixed with 2.5% glutaraldehyde in 0.2 mol/L phosphate-uffered saline solution (PBS; pH 7.4). Specimens postfixed in 1%smium tetroxide, were dehydrated and dried in hexamethyldisi-
azane (Sigma) before coating with gold palladium and examina-ion in a Cambridge Stereoscan 260 SEM. We also used standardechniques for specimen preparation for TEM. Cells grown intandard culture dishes were subjected to Karnofsky fixative andostfixed in 1% osmium tetroxide before dehydration and embed-ing in Eponate resin (Ted Pella, Redding, CA, USA). Specimenstained with lead citrate and uranyl acetate were examined in ahilips TECNAI10 TEM.17,18
ell Density and Growth Curve
mmortalized hepatocytes were inoculated into 6-well plates at annitial density of 1.5 � 104 cells/well in 2.0 mL DMEM containing0% fetal bovine serum were incubated at 37°C with 5% CO2. Theells were not passaged during the 7-day experimental periods. Cellumbers calculated after trypsinization (0.25%) and stained with.4% trypan blue after 24 hours of culture. The cell growth curveas drawn with culture time as the abscissa and cell numbers as therdinate. Primary cultured porcine hepatocytes were used as theontrol group. Each cell count was performed on 2 wells.
easurement of Albumin and Urea Productions
e determined porcine albumin production in supernatants ofmmortalized porcine hepatocytes by using an enzyme-linkedmmunosorbent quantitation kit (Bethyl, Montogomery, Tex,
SA). As described in detail elsewhere,19 a polyclonal antibodygainst porcine albumin, peroxidase-conjugated goat anti-rabbitlbumin, and purified porcine albumin as the standards. Toxamine urea synthesis by immortalized porcine hepatocytes,rea concentrations in collected media were quantitated by aommercially available kit (Sigma). All assays were performedccording to the manufacturer’s instructions.
estern Blot Analysis
ells were lysed with buffer containing 20 mmol/L PBS (pH 7.4),% NP40 (Sigma), 0.5% sodium deoxycholate, 0.1% sodiumodecyl sulfate (SDS), and 0.01% protease inhibitor cocktailNovagen, USA). After cell debris was removed by centrifugationt 13,000 rpm for 5 minutes, the supernatant was used as theample, with 20 �g of protein extract dissolved in sample bufferontaining 140 mmol/L Tris (pH 6.8), 22.4% glycerol, 6% SDS,0% mercaptoethanol, and 0.02% bromphenol blue. After boiling,he sample was loaded onto a 10% polyacrylamide gel for electro-horesis using a Mini-Protean-3-electrophorese-cell (Bio-Rad,ercules, CA, USA). Proteins were transfered to polyvinylideneuoride membranes. The following primary antibodies were used:ouse monoclonal anti–SV40 LT antibody (Santa Cruz, CA,SA), mouse monoclonal anti-hTERT (Santa Cruz), and mouse
nti-human CK18 (Santa Cruz). Bound antibody was detected
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sing horseradish peroxidase–conjugated secondary antibodiesnd an enhanced chemiluminescence detection system (Biologyndustries, Israel).
everse-Transcription Polymerase Chain Reaction Analysis
otal cellular RNA was isolated from primary porcine hepatocytesnd immortalized porcine hepatocytes at passage 60 by using arizol kit (Invitrogen). Reverse transcription polymerase chain
eaction (RT-PCR) was performed using the One-Step RT-PCReagent kit (Invitrogen) according to the manufacturer’s protocols.riefly, the first program of complementary DNA (cDNA) synthe-
is was performed for 1 cycle at 45°C for 30 minutes, and thenubjected to 30 cycles of amplification, followed by a final extensionor 5 minutes at 72°C. PCR amplification was performed using theollowing primer sequences: SV40 LT-1 (5=-CAAATGTGGTAT-GCTGAT-3=) and SV40 LT-2 (5=-GGCTATGGGAACTGGA-
=) for SV40 LT, hTERT-1 (5=-GT ACT TTG TCA AGG TGGTG TGA-3=) and hTERT-2 (5=-GG CTG GAG GTC TGTAA-3=) for hTERT, ALB-1 (5=-TGTGTTGCTGATGAGT-AGC-3=) and ALB-2 (5=-GAGACAGAGTAACGACGACT-3=)
or albumin,20 CYP-1 (5=-CAGTAAGAAGCACAAGGA-ACCA-3=) and CYP-2 (5=-TGAAGGAGAAGTTCTGCAGG-3=)
or CYP3A29 of porcine cytochrome P450,21 GAPDH-1 (5=-CTTCATTGACCTCCACTACAT-3=) and GAPDH-2 (5=-CAAAGTTGTCATGGATGACC-3=) for GAPH20 as a control.he PCR products were separated on 1% agarose gel containingthidium bromide, and the bands were viewed under ultravioletight.
iazepam Metabolism
o assess its metabolism, we analyzed diazepam clearance onmmortalized porcine hepatocytes during the 5-day experimentaleriods. Immortalized porcine hepatocytes were suspended in 2L DMEM containing diazepam (50 �g/mL) at 37°C in a 5% CO2
ncubator. At designated times, collected medium samples weretored at �80°C for analysis. Diazepam was quantified by high-erformance liquid chromatography as described elsewhere.22,23
e performed all tests, including controls, at least in triplicate.
umorigenicity Assay
o examine the potential of the immortalized porcine hepatocyteell line to form tumors in vivo, 1 � 106 cells were injectedubcutaneously into 8-week-old nude mice. As controls, nude miceeceived subcutaneous transplantation of 1 � 106 HepG2 humanransformed liver cells. Animals were examined for the presence ofumors weekly over a 6-month period.
ransplantation Experiments
e divided Lewis rats in the transplantation experiments into theollowing groups: group 1 (G1), intrasplenic transplantation of 3.0 �07 immortalized porcine hepatocytes (n � 8); group 2 (G2), intra-plenic transplantation of 3.0 � 107 primary porcine hepatocytesn � 8); and group 3 (G3), intrasplenic injection of 0.5 mL DMEMedium (n � 8). All animals underwent 90% hepatectomy at 24
ours after transplantation and received daily intramuscular injec-ions of FK506 (1 mg/kg) to suppress immune rejection of xeno-eneic hepatocytes. The 90% hepatectomy has been described inetail an elsewhere.3,24 We observed survival and blood biochem-
cal parameters. o
tatistical Analysis
uantitative results were presented as mean values � SD. Statis-ical differences were determined by the Mann-Whitney U test,ollowed by a 2-tailed Student t test. Survival time was analyzedsing the Kaplan-Meier method and significance tested using the
og-rank test. A P value of �.05 was considered to be statisticallyignificant.
ESULTSstablishment of Immortalized Porcine Hepatocytes
rimary porcine hepatocytes were transfected with theecombinant retroviral vectors by exposure to amphotropicetroviral supernatants. The transfected clones grew withinweeks, whereas the nontransfected primary porcine hepa-
ocytes died within 2 weeks. After culturing for 2 years initro, all 3 transfected clones continued to proliferate. Onef them, referred to as HepLi cells, was composed of cellsaving large round nuclei with a few nucleoli and manyranules in the cytoplasm under phase-contrast microscopyFig 1A and 1B). SEM and TEM showed that HepLi cellsaintained a round nucleus with nucleoli, moderate num-
ers of mitochondria, and numerous rough endoplasmiceticula (Fig 1C and 1D).
nalysis of SV40 LT and hTERT
o determine the successful transfection and translationnto protein of the SV40 LT and hTERT, we detected theirxpression in immortalized cells by using Western blots (FigA). Moreover, the expression of SV40 LT and hTERTRNA determined by RT-PCR indicated successful trans-
ection of the 2 exogenous genes in the HepLi cells (FigB).
ell Number and Growth Curve
e analyzed the growth kinetics of HepLi cells undertandard culture conditions, calculating cell numbers byaily determinations with trypan blue exclusion methods.epLi cells cultured in DMEM proliferated from 1.5 � 104
o 2.1 � 105 cells/well over 7 days, revealing a significantlyncreased number of HepLi cells throughout all observationeriods (Fig 3A). In contrast, primary cultured hepatocytesid not proliferate over 7 days. To date, the HepLi cellsave been maintained for �250 passages with an average ofopulation doubling every 6–8 hours.
lbumin Secretion and Urea Production
o further monitor synthesis functions of immortalizedorcine hepatocytes, we measured their albumin secretionnd urea production. Table 1 shows a steady increase inlbumin and urea concentrations in culture media over 9ays.
ene Expression of Liver-Enriched Functionsn HepLi Cells
igure 2B shows the gene expression of hepatocyte markers
f porcine albumin mRNA by RT-PCR in HepLi cells.
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T-PCR results showed that HepLi cells also expressedYP450 mRNA (Fig 2B). HepLi cells and primary porcineepatocytes also showed expression of porcine GAPDHRNA (Fig 2B).
xpression of Cell-Type Specific Protein
o confirm the epithelial origin of immortalized porcineepatocytes, we evaluated the expression of cytokeratin-18
n HepLi cells. The results of Western blot analysis revealed
A
C
ig 1. Morphologic characteris-ics of immortalized porcine hepato-ytes. The morphology of immortal-
zed porcine hepatocytes in cultureas demonstrated by phase con-
rast microscopy. (A) �200; (B) �40.he morphology of immortalizedorcine hepatocytes was also per-
ormed by transmission electron mi-roscopy (TEM) and scanning elec-ron microscope (SEM). (C) TEM6,200; (D) SEM �608.
ig 2. Expression of immortalizing genes and porcine liver-speclotting and/or reverse-transcription polymerase chain reactionenes were expressed in HepLi cells and not detected in primarxpression of cytokeratin-18 in both HepLi cells and primaryxpression of hepatocyte markers of porcine albumin and cytoc
mmortalizing genes SV40 LT and hTERT in HepLi cells. GAPDH was
hat cytokeratin-18 was consistently expressed in bothepLi cells and primary porcine hepatocytes (Fig 2A).
iazepam Metabolism
e incubated diazepam with immortalized porcine hepato-ytes on various postculture days. Figure 3B shows the de-rease in diazepam concentration. This result showed stabilityf diazepam metabolic activity by immortalized porcine hepa-ocytes.
B
D
enes in immortalized porcine hepatocytes detected by WesternCR). (A) Western blotting shows that both SV40 LT and hTERTcine hepatocytes; the result of Western blotting also shows theine hepatocytes. (B) The results of RT-PCR show the mRNAe P450 genes in HepLi cells and the mRNA expression of both
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valuation of Tumorigenicity of HepLi Cells
e investigated the tumorigenic potential of the immortal-zed porcine hepatocyte line in nude mice. When injectedubcutaneously with 1 � 106 HepLi cells, the nude miceemained tumor free for as long as 6 months, whereas nudeice transplanted with the same amount of HepG2 cells
eveloped neoplasms within 3–4 weeks after injection.
ffect of Hepatocyte Transplantation on Liverunction and Survival
e evaluated the therapeutic effect of immortalized por-ine hepatocytes in a rat model of ALF induced by 90%epatectomy. Biochemical parameters, including total bili-ubin (Fig 4A), ammonia (Fig 4B), and prothrombin timeFig 4C), increased gradually to 3 days after 90% hepatec-omy in G3 rats. In contrast, 90% hepatectomy rats trans-lanted with either immortalized porcine hepatocytes orrimary porcine hepatocytes showed significant improve-ent in these parameters if the animal did not die due toLF (P � .05 for G1 or G2 vs. G3). As shown in Fig 4D,
ats transplanted with immortalized porcine hepatocytes orrimary porcine hepatocytes showed better survival com-ared with G3 rats (P � .01 for G1 or G2 vs. G3).
ig 3. Growth curve of HepLi cells and diazepam metabolism oore rapidly than primary porcine hepatocytes (PPH) during aostculture days.
Table 1. Albumin and Urea Production by HepLi Cells Culturedon the Single Layer of Collagen Gel
ays in CultureAlbumin Production
(ng/mL)Urea Production
(�g/mL)
1 28 683 46 1195 63 2417 97 315
a9 136 359
ISCUSSION
e have described the preparation of immortalized porcineepatocyte cell lines by double transfection with SV40 LTnd hTERT genes. The HepLi cells exhibited active func-ional characteristics of primary porcine hepatocytes. Fur-hermore, intrasplenic transplantation of immortalized por-ine hepatocytes significantly improved liver function androlonged the survival rate of rats induced with ALF by0% hepatectomy.At least 2 barriers, namely, replicative senescence and
risis, constrain the proliferative capacity of primary cells inulture.25,26 Expression of SV40 LT enables cultured cellso bypass replicative senescence, and ectopic expression ofTERT circumvents crisis. hTERT appears to be essentialor immortalization.27–29 Another study has shown thatmmortalization with SV40 LT plus hTERT was much morefficient than hTERT alone.30 Therefore, in the presenttudy, we cotransfected both SV40 LT and hTERT genesnto primary porcine hepatocytes to establish the immortal-zed porcine hepatocyte cell lines. Our results agree witharlier studies27,29 that reported cotransfection of SV40 LTnd hTERT genes to be an effective method to immortalizenumber of cell types.The present study demonstrated that the newly immor-
alized porcine hepatocyte cell line showed characteristicsimilar to primary porcine hepatocytes. The RT-PCR anal-sis showed that HepLi cells exhibited a number of liver-nriched functional markers of primary porcine hepato-ytes, including expression of porcine albumin and porcineytochrome P450 (CYP) mRNA. Western blot analysis ofepLi cells suggested that these cells also expressed SV40T, hTERT, and cytokeratin-18.Cytochrome P450 plays an important role in most drugetabolism. Diazepam has been used to evaluate CYP
Li cells. (A) Cell growth curve shows that HepLi cells grew muchy period. (B) Diazepam metabolism by HepLi cells on various
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ctivities. Pigs possess the main human enzyme of drug
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iotransformation (CYP3A),31 and diazepam metabolisman be achieved by porcine hepatocytes.23,32 In the presenttudy, Fig 2B shows the mRNA expression of CYP3A29 ofhe CYP system. Figure 3B shows diazepam metabolism bymmortalized porcine hepatocytes. Taken together, theseesults suggest that the immortalized porcine hepatocytesetained the metabolic functional characteristics of primaryorcine hepatoyctes.Albumin secretion and urea production by porcine hepa-
ocytes were characterized as markers of other liver-specificunctions. In this study, both albumin and urea production
ig 4. Intrasplenic transplantation of HepLi cells in a rat mepatectomy, the levels of total bilirubin (A) and ammonia (B
ntrasplenic transplantation of 3.0 � 107 HepLi cells (n � 8); gepatocytes as a positive control (n � 8); and group 3 (G3), i
n � 8). The G3 rats experienced a rapid increase in total bilirubinn G1 and G2 showed significant improvement in these parammmortalized porcine hepatocytes or primary porcine hepatocyte2 vs. G3).
y immortalized porcine hepatocytes showed stable in- r
reases over the 9-day period (Table 1). Continuous albu-in production of immortalized porcine hepatocytes can be
ccepted to be a marker to identify hepatocytes. Moreover,rea synthesis is used frequently for the detoxification ofmmonia as hepatocytes.
The continuous expression of high levels of SV40 LT mayause chromosomal abnormalities that result in malignantransformation.13,33 We decided to test whether HepLi cellsould cause tumors in nude mice. Nude mice inoculatedith HepG2 neoplastic cells developed tumors after 3eeks. In contrast, nude mice inoculated with HepLi cells
of acute liver failure (ALF). After ALF was induced by 90%prothrombin time (C) are shown in 3 groups: group 1 (G1),
2 (G2), intrasplenic transplantation of 3.0 � 107 primary ratsplenic injection of 0.5 mL DMEM medium as a negative controlonia, and prothrombin time. In contrast, 90% hepatectomy rats
s (P � .05 for G1 or G2 vs. G3). (D) Rats transplanted withd better survival rates compared with G3 rats (P � .01 for G1 or
odel) androupntras, amm
eters ha
emained tumor free after 6 months, a finding consistent
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IMMORTALIZED PORCINE HEPATOCYTES 1905
ith earlier studies showing that expression of SV40 LT andTERT was not sufficient to produce oncogenic transfor-ation of primary human cells.34–36
Because of the critical shortage of donor livers forransplantation, xenogeneic hepatocytes represent an alter-ative for the treatment of acute liver failure. Hepatocyteenotransplantation has been performed successfully innimal models of acute liver failure. Some researchers havelready obtained remarkable results wherein rats with he-atocyte xenografts survived longer than those animals thatid not receive hepatocyte xenotransplantation.5 Moreover,notransplanted porcine hepatocytes have been shown tomprove liver function and to prolong the survival of pigsith liver failure induced by D-galactosamine treatment.37
n the present study, we also showed that immortalizedorcine hepatocytes could support rats induced with 90%epatectomy. Our immortalized porcine hepatocyteschieved a remarkable degree of protection, improving liverunctions among rats with ALF. In addition, the survival ofats transplanted with immortalized porcine hepatocytesas significantly different from that of rats transplantedith DMEM medium, suggesting that transplanted immor-
alized porcine hepatocytes were responsible for their ther-peutic effect. On an encouraging note, researchers haveeported the first successful xenotransplantation of primaryorcine hepatocytes in nonhuman primates.6 In the nearuture, immortalized porcine hepatocytes, which maintainhe advantage of infinite proliferation capacity, may beotentially useful in xenotransplantation.In summary, we generated immortalized porcine hepato-
yte cell lines by cotransfecting SV40 LT and hTERT intorimary porcine hepatocytes. The phenotypes of the im-ortalized porcine hepatocytes were similar to those of
rimary porcine hepatocytes. Intrasplenic transplantationf immortalized porcine hepatocytes significantly improved
iver functions and prolonged survival after induction ofLF in rats. This generation of immortalized porcineepatocytes might prove to be helpful for xenotransplanta-ion.
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