Table 1. Overview of the functionality and immortalization strategy of in vitro established human and rodent hepatic cell lines.

A1AT, αl-antitrypsin; AFP, α-fetoprotein; AhR, aryl hydrocarbon receptor; ALB, albumin; ALF, acute liver failure; A2M, α2-macroglobulin; APO, apolipoprotein; Arnt, AhR nuclear translocator; ASGP(R), asialoglycoprotein (receptor); BCRP, breast cancer resistance protein; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog; BSEP, bile salt export pump; CAR, constitutive androstane receptor; C/EBP, Ccaat-enhancer-binding protein; CD, cluster of differentiation; CK, cytokeratin; CLDN, claudin; CYP, cytochrome P450; DMSO, dimethyl sulphoxide; DPP, dipeptidyl peptidase; EH, epoxide hydrolase; EPCAM, epithelial cell adhesion molecule; GGT, γ-glutamyltranspeptidase; G6P, glucose-6-phosphate; GPX, gluthatione peroxidase; GS, glutamine synthetase; GST, gluthatione S-transferase; HBCF, human blood coagulation factor; HGFR, hepatocyte growth factor receptor; HNF, hepatocyte nuclear factor; HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase; IL, interleukin; INF, interferon; MDR, multidrug resistance protein; mRNA, messenger ribonucleic acid; MRP, multidrug resistance-associated protein; NADPH, nicotinamide adenine dinucleotide phosphate; NCAM, neural cell adhesion molecule; NTCP, sodium taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; PEPCK, phosphoenolpyruvate carboxykinase; PT, prothrombin; PXR, pregnane X receptor; Rb, retinoblastoma; SCID, severe combined immunodeficiency; SOD, superoxide dismutase; SV40 Tag, simian virus 40 large T antigen; TAT, tyrosine aminotransferase; TF, transferrin; UGT, uridinediphosphate-glucuronosyltransferase.

Human hepatic cell lines
Cell line	Immortalization strategy	Functionality	Ref
Fa2N-4	Adult hepatocytes	- Possessed, in comparison with cryopreserved human hepatocytes: ■ Significantly lower basal expression level of the nuclear receptor CAR and several drug metabolizing enzymes and transporters, namely CYP1A2/2D6/2E1/1A1, UGT1A1/1A6/2B15/2B4, sulfotransferase, NTCP, OCT1, OATP1B1/1B3, MRP2 and BSEP. ■ Markedly higher MDR1 mRNA levels. ■ Similar basal expression of BCRP, PXR and AhR. ■ Apparently higher expression of most transcription factors and coactivators/corepressors that have been associated with PXR and CAR mediated enzyme induction. -Were incapable of metabolizing compounds due to low basal levels of drug-metabolizing enzymes. -Exhibited, at early passage, inducible CYP1A2/2C9/3A4, UGT1A and MDR1 mRNA levels as well as CYP1A2/2C9/3A4 activities and could distinguish inducers from non-inducers. At higher passages, the cells lost the ability to induce. -Were not tumorigenic.	[22, 86, 176]
	Transfection
	SV40 Tag
FH-TERT	Fetal hepatocytes	-Expressed CYP450 mRNA and maintained, in contrast to passaged fetal hepatocytes, liver-enriched differentiation markers, especially C/EBPα and HNF4 as well as elevated levels of HGFR. -Possessed glycogen storage and G6P activity, in a pattern similar to primary fetal hepatocytes. -Produced urea and retained level of ALB synthesis equivalent to HepG2 cells. -Displayed no in vitro anchorage-independent growth or in vivo tumor formation. -4 weeks after intrahepatic transplantation in immunodeficient mice, FH-hTERT engrafted, survived and expressed ALB, A1AT, and TF mRNA levels comparable with primary human hepatocytes. These experiments illustrated the ability of FH-hTERT to differentiate into mature hepatocytes and to display significant hepatocellular gene expression. ➢Culture conditions used in these studies were designed at supporting cell proliferation, and conditions have not been optimized for inducing differentiated hepatocellular functions.	[2]
	Retroviral vector
	hTERT
Hc3716- hTERT	Fetal hepatocytes	-Maintained normal mammalian cell morphology. -Exhibited protein expression of ALB, CK8 and CK18, but not AFP. ALB levels were higher than in control, passaged Hc3716 cells. -Possessed inducible CYP3A4/7 mRNA levels. -Exhibited wild-type p53 responsiveness. -Did not show typical oncogenic phenotype traits. ➢In this study, it is shown that inappropriate culture conditions induce senescence programming in human cells. Adapting the culture medium allowed the human fetal hepatocytes to extend their lifespan over 80 passage doublings, instead of 10 in normal culture conditions.	[108]
	Retroviral vector
	hTERT
HepLi5	Adult hepatocytes	-Expressed HBCF-X, GS, GST, ALB and CYP450 mRNA. -Retained ALB secretion and urea production, though at low levels compared to primary hepatocytes. -Dislayed CYP1A2 activity. -Were not tumorigenic. -Possessed significantly enhanced cellular functions after large-scale culture in roller bottles.	[141]
	Retroviral vector
	SV40 Tag
HepLL	Adult hepatocytes	-Displayed morphologic characteristics of liver parenchymal cells. -Expressed HNF4, HBCF-X, GST-n and ALB mRNA as well as ALB and CYP2E1 protein but no ASGP mRNA. -Stained positive for human hepatocyte special antigen but negative for AFP. -Secreted ALB and urea at levels not significantly different from primary cultured human hepatocytes. -Synthesized glycogen. -Were not tumorigenic after transplantation into SCID mice. -Possessed a good potential of regeneration and active metabolic function in recipient organs.	[83]
	Lipid mediated gene transfer (lipofectamine reagent)
	SV40 Tag
HepZ	Adult hepatocytes	-When grown in bioreactor, cells were able to secrete ALB and A2M and possessed inducible CYP450 activity. ➢pRB andp53 constructs under control of ALB promoter.	[112]
	Lipid mediated gene transfer (lipofectamine reagent)
	Antisense constructions for Rb and p53 + Cotransfection of E2F transcription factors and cyclin D1
HHE6E7T-1/2	Small hepatocytes	-Displayed epithelial-like morphology. -Retained characteristics of differentiated hepatocytes, though functions such as ALB secretion as well as mRNA expression levels of ALB, HNF4 and A1AT decreased gradually as the passages progressed. CK18 mRNA levels were detected throughout the culture period and no AFP expression was observed. -Were positive for vimentin staining. -Showed chromosomal instability after long-term passage. -Exhibited no tumorigenic properties after transplantation in SCID mice. -Improved survival of acetaminophen-induced ALF mice through possible redifferentiation in vivo. It was suggested that once transplanted, cells may support ammonia metabolism and gluconeogenesis, even though cells at latter passages did not possess ureogenesis or gluconeogenesis. ➢In this study, transduction of hTERT alone could not extend the life span of normal human adult hepatocyte.	[20, 153]
	Lentiviral and retroviral vectors
	HPV16 E6/E7 + hTERT
HHL(−5/−7/−16)	Adult hepatocytes	-Contained markers of hepatocyte and biliary phenotype(CK7/8/18/19). -Expressed CYP450 protein at levels comparable to Huh-7 and HepG2 cells. -Produced ALB, though at lower levels than Huh-7 and HepG2 cells. -Stained negative for AFP and did not display elevated nuclear expression of p53 protein. -Possessed active gap junctions. -Respond to INF- α stimulation by upregulation of major histocompatibility complex I and II -Exhibited, in contrast to the Huh-7 and HepG2 cells, increased capacity to bind recombinant hepatitis C virus-like particles.	[19]
	Retroviral vector
	HPV16 E6/E7
IHH-A5	Adult hepatocytes	-Were morphologically and functionally more similar to hepatoma cell lines than primary hepatocytes in culture. -Secreted different plasma proteins, including ALB, APO-B and fibrinogen at relatively high rates, within the range observed for early primary human hepatocyte cultures. Addition of IL-6 to the culture medium resulted in increased fibrinogen secretion and decreased ALB production, demonstrating a proper acute-phase response. -Produced detectable amounts of APO-A1. -Exhibited bile-canalicular structures that, in some cases, accumulated the organic anion glutathione-methylfluorescein. Cell cultures were partly polarized and expressed the efflux transporters, MDR1 and MRP1, on the membranes of apical vacuoles or on the lateral membranes of adjacent, proliferating cells, respectively. -Did not maintain active Na+ -dependent bile salt uptake. -Displayed similar lipoprotein metabolism as HepG2 cells.	[135]
	Lipid mediated gene transfer (lipofectin reagent)
	SV40 Tag
NeHepLxHT	Neonatal hepatocytes	-Displayed characteristic morphology of primary fetal liver cells. -Maintained epithelial characteristics as evidenced by immunostaining for epithelial cell markers, the cytokeratins. -Possessed gene expression profile similar to human neonatal hepatocytes, with positive expression of A1AT, CKIT, CLDN3, EPCAM, NCAM mRNA and no detection of AFP, ASGPR or CYP3A4. The very low ALB mRNA levels compared to HepG2 cells and the expression of CK19 in early passages, indicated the progenitor nature of the cells. -Retained a normal diploid karyotype.	[9]
	Retroviral vector
	hTERT
OUMS-29	Fetal hepatocytes	-Displayed epithelial morphology. -Maintained gene expression of ALB, ASGPR, bil-UGT, GS, GST-Π, HBCF-X, AhR and Amt. -Secreted ALB, AFP, TF, A1AT and APO A-1. -Possessed inducible CYP1A1/2 mRNA levels and activity. -Displayed chromosomal abnormalities. -Protected 90% hepatectomized rats from hyperammonemia and prolonged their survival after intrasplenic transplantation. ➢Overexpression of HNF4α2 led to development of OUMS-29/H-H cell line with increased liver-specific gene expression, such as A1AT, apolipoproteins, HBCF-X and HNF1 α.	[21, 122, 136]
	Lipid mediated gene transfer (lipofectin reagent)
	SV40 Tag
PH5CH	Adult hepatocytes	-Displayed epithelial appearance. -Expressed human CK and ALB protein. -Possessed low colony-forming efficiency. -Were not tumorigenic.	[137]
	Lipid mediated gene transfer (lipofectin reagent)
	SV40 Tag
THLE-2/-3	Adult hepatocytes	-Displayed epithelial morphology. -Secreted ALB and expressed CK18, TF, A1AT, A2M, GST-Π and very low levels of GGT at early passages. CK19 expression could only be determined at later passages. Cells were uniformly negative for AFP and factor VIII. The appearance of CK19 and decreased ALB secretion at later passages demonstrated that cells undergo dedifferentiation in culture. -Retained mRNA expression of phase II enzymes such as EH, catalase, GPX, SOD and GSTs at levels comparable to human liver, with GST-Π and α mRNA as the dominant form in THLE cells or human liver, respectively. -Maintained NADPH CYP reductase expression at a lower steady-state mRNA level than in human liver. -Were able to metabolize three carcinogens, which suggested the presence and activity of CYP1A2/1A1, CYP2E1 and CYP3A4. However CYP1A2, CYP2E1, CYP3A4, CYP2A3 and CYP2D6 mRNA were not detected. The steady-state mRNA levels of CYP1A1 increased after exposure to Aroclor 1254 or B[ α ] P. -Displayed chromosomal abnormalities. -Possessed no tumorigenic properties. ➢Besides the use of viral vectors, strontium phosphate transfection was also applied (THLE-1 cells). Immortalization could only be reproducibly established by retroviral transduction.	[104]
	Retroviral vector
	SV40 Tag
TPH1	Adult hepatocytes	-Exhibited altered cell morphology resembling low-differentiated epithelial cells. -Expressed no A1AT or AFP mRNA. -Secreted ALB. -Possessed G6P activity. -Reactivated telomerase immediately after senescence. -Displayed anchorage-independent growth at latter stages, providing evidence for transformed phenotype.	[113]
	Strontium phosphate precipitation
	HCV core gene
Conditional immortalization
16T-3	Adult hepatocytes	Reverted 16T-3 cells: -Showed enhanced mRNA levels of transcriptional factors, C/EBP α and HNF4 α as well as increased mRNA expression of hepatocyte-specific genes, including ALB, GST-Π, HBCF-X, bil-UGT, CYP3A4, GS and ASGPR. -Possessed increased ALB production and lidocaine metabolism, though at lower levels than normal human hepatocytes. -Intraportal transplantation in a pig model of ALF induced by D-galactosamine recovered TBL, AST, NH and PT levels and prolonged survival without tumor formation.	[111]
	Retroviral vector
	hTERT
	Tamoxifen-mediated selfexcision (Cre-LoxP)
cBAL111	Fetal hepatocytes	-Expressed relatively high mRNA levels of immature markers, GST-Π and AFP, and very low mRNA levels of mature markers, ALB, A1AT and TF. Transcript levels of HNF4 α increased after prolonged culturing. -Stained positive for GS, ALB, CK18, CK19, vimentin and the progenitor cell marker CD146 but displayed CK18 in a pattern characteristic of dedifferentiated human hepatocytes. -Produced urea and ALB, though at lower levels than mature human hepatocytes. -Retained no CYP1A2 & 3A4 activity (no elimination of lidocaine) but were able to eliminate galactose. -Displayed no anchorage-independent growth. -Possessed the ability to differentiate into functional hepatocytes once transplanted in vivo, without the occurrence of tumor formation. ➢cBALUl cells resembled cells with progenitor characteristics rather than fully differentiated hepatocytes. However, there was a trend of increased and decreased expression of mature and immature markers, respectively, with culture time.	[7]
	Lentiviral vector
	hTERT
	Transcriptional regulation (Tet-on approach)
HepCL	Fetal hepatocytes	-Displayed morphological characteristics of liver parenchymal cells. -Stained positive for ALB, CK18 and CK19. -Produced amounts of ALB and urea comparable to those of unmodified primary human fetal hepatocytes. -Were not tumorigenic after transplantation into SCID mice. -Transplanted cells rescued mice after 90% hepatectomy, produced ALB and CK18 and were superior to HepG2 cells regarding metabolic support during ALF.	[124]
	Retroviral vector
	SV40 Tag
	Temperature-based regulation
HepLi-4	Adult hepatocytes	Reverted HepLi-4 cells: -Expressed similar GS and somewhat lower UGT1A1 mRNA levels than adult human liver. ALB and GST-Π mRNA levels were extremely lower or higher, respectively, compared to the human liver. This indicates that HepLi-4 cells are not fully differentiated after reversion. -Prolonged the survival of common bile duct ligated mice after intrasplenic transplantation. -Were not tumorigenic.	[87]
	Retroviral vector
	SV40 Tag
	Tamoxifen-mediated selfexcision (Cre-LoxP)
HLTC-7/-11/-15/-17/-19	Adult hepatocytes	-Grew as islands or sheets of cuboidal cells (HLTC-17) or displayed a more dispersed cuboidal-elongated morphology (HLTC-7/-11/-15/-19). -Secreted fibrinogen at fairly constant rate in all tested cell lines at permissive (33,5°C) and non-permissive (39,5°C) temperature. -Exhibited no ALB, AFP, A1AGP or PT secretion in any cell line at both temperatures. -Cell lines HLTC-7,-15 and -19, produced A1AT at permissive temperature. However, at non-permissive temperature the secretion of A1AT was upregulated or became detectable in all the cell lines. -Cell lines HLTC-17 and -11 possessed no CYP activity at any temperature even after induction and stained positive for ALB, CK18, CK7, CK19 and vimentin, but negative for CK8, with almost identical patterns at both temperatures. -HLTC-17 cells showed considerable aneuploidy with chromosomal rearrangements. All HLTC cells contained a derivative chromosome with loss of the short arms of chromosomes 11 and 12. -Displayed no anchorage-independent growth. ➢The results indicated progressive phenotypic instability and loss of differentiated functions. Conversion to the non-permissive temperature did only allow significant expression of a limited repertoire of differentiated functions by the immortalized human hepatocytes.	[103]
	Retroviral vector
	SV40 Tag
	Temperature-based regulation
IHH10(.3)/12	Adult hepatocytes	-Displayed morphology reminiscent of differentiated hepatocytes. -Expressed ALB, A1AT, ASGPR and CYP450 mRNA levels. -Secreted liver-specific proteins, ALB and fibrinogen, at levels similar to Huh-7 cells but lower than primary hepatocytes. The IHH12 cell line did only produce fibrinogen after de-immortalization, suggesting the acquirement of a higher differentiation status in this setting. However, Cre-recombinase treatment of IHH12 cells did not significantly improve the production of ALB. -Possessed inducible CYP1A1/2 activity. -Were not tumorigenic and rescued mice from lethal doses of acetaminophen. ➢Combination of immortalizing genes hTERT & Bmi-1 was insufficient to immortalize non-dividing human hepatocytes.	[23]
	Lentiviral vector
	SV40 Tag + hTERT (IHH10) or SV40 Tag + hTERT + Bmi-1 (IHH12)
	Recombinase- based control (Cre-LoxP)
NKNT-3	Adult hepatocytes	-Displayed morphological characteristics of liver parenchymal cells and looked more differentiated after reversion. -Expressed bil-UGT, GS and GST-Π mRNA levels, which increased substantially after reversion. Contradicting results were published regarding expression of ALB and HBCF-X mRNA levels. One paper demonstrated that ALB and HBCF-X mRNA were newly introduced in the reverted cells whereas several other papers already reported expression of these genes and ASGPR mRNA in non-reverted cells. Nevertheless, although reversion did stimulate differentiation, mRNA levels of ALB, A1AT and TF were maximally 0,1% of primary human hepatocytes. -Were not tumorigenic after transplantation into SCID mice and both NKNT-3 and reverted NKNT-3 significantly improved biochemical parameters in transplanted rats, protecting as such 90% hepatectomized rats from hyperammonemia and prolonged their survival. ➢Additional experiments revealed that introduction of p21 into human immortalized hepatocytes can increase ALB expression and induce a differentiated morphology.	[6, 85, 136, 140, 152]
	Retroviral vector
	SV40 Tag
	Recombinase- based control (Cre-loxP)
YOCK-13	Adult hepatocytes	-Displayed morphological characteristics of normal human hepatocytes. -Expressed markers of hepatocytic differentiation including ALB, ASGPR, bil-UGT, CYP3A4, GS, GST-Π, and HBCF-X. -Immortalized and reverted cells possessed no tumorigenic properties in SCID mice. -Xenotransplantation in totally pancreatectomized pigs, decreased hyperglycemia and prolonged survival without adverse effects such as portal thrombosis, liver necrosis, pulmonary embolism and tumor development. ➢The YOCK-13 hepaticcell line is derived from the reversible immortalized human hepatic cell line, TTNT-16-3, by co-expression of modified insulin.	[110]
	Retroviral vector
	hTERT
	Tamoxifen-mediated self-excision (Cre-LoxP)
Rodent hepatic cell lines
Cell line	Immortalization strategy	Functionality	Ref
AdPX3/4	Rat adult hepatocytes	-Secreted ALB at early passage, though at lower levels than primary cultured hepatocytes and many CWSV cell lines. Moreover, ALB amounts declined with continued passage in culture. The cells also produced additional plasma proteins, including TF, hemopexin and C3 complement but no AFP. -AdPX4 cells were, at least at low passage, not tumorigenic. ➢Use of E1A or E1B gene expression alone was insufficient to induce hepatocyte proliferation. ➢Transformation frequencies were enhanced when growth factors were added to the culture medium.	[25]
	Calcium phosphate precipitation
	E1A & E1B
C3-II-B-2-3 C4-1-B-2 C8-IV P9 SV40RH1	Rat adult hepatocytes	-Produced ALB (SV40RH1/C3-II-B-2-3/C4-1-B-2/C8-IV). -Possessed bil-UGT activity (SV40RH1/P9). -Were able to resynthesize gluthatione from methionine and cysteine (SV40RH1/P9). -Expressed low levels of phase II enzymes, GGT and GST-Π (SV40RH1/P9). -Contained cells expressing vimentin. The SV40RH1 was the only cell line in which vimentin-negative cells were present.	[133]
	Calcium phosphate precipitation
	SV40 DNA
CWSV	Rat adult hepatocytes	-Were derived from colonies of ALB-producing epithelial cells, which were morphologically more similar to established hepatoma cell lines than cultured primary hepatocytes. -At low passage, the cell lines, CWSV1, 2, 14, 16 and 17 expressed high ALB mRNA levels and possessed TF, A1AT and PEPCK mRNA levels which were similar to the liver but higher than to 2 hepatoma cell lines (H4IIEC3 & McA-RH7777). At higher passage ALB expression decreased only slightly in the CWSV1, 2 and 17 cell lines but markedly in CWSV14 and 16 cells. TF levels were marginally diminished in all cell lines whereas A1AT and PEPCK were only highly maintained in CWSV1 and 14 cells, respectively. No AFP expression could be detected throughout cultivation, except in high passaged CWSV14 cells. -Exhibited variable ALB production, ranging from high (CWSV1, 2, 9, 10, 14, 15, 16 and 17) to low (CWSV4 and 5) levels. CWSV8 cells did not produce detectable ALB amounts from passage 11 onwards. ALB-producing cell lines also secreted other plasma proteins, including, TF, hemopexin and C3, but no detectable amounts of AFP. -CWSV1, 2, 16 cells were only tumorigenic at higher passages whereas transplanted CWSV 14 and 17 cells already formed tumors at low passages. ➢Transformation frequencies were enhanced when growth factors were added to the culture medium. ➢It was shown that culture conditions used for transformation could greatly influence the differentiated hepatic phenotype of transformed cells. For example formation of ALB secreting colonies was greatly increased after addition of DMSO to the culture medium. ➢Experiments demonstrated that ALB expression was apparently temperature-independent.	[105, 134, 177]
	Calcium phosphate precipitation
	SV40 DNA
RH(1-4/6-10)	Rat adult hepatocytes	-Displayed morphology consistent with that of immature hepatocytes and identical to certain cultured undifferentiated hepatoma cells. -Did not express CK19 protein levels. -Retained ALB-positive cells, but mRNA and protein expression were weaker than in rat hepatocytes. -Showed AFP protein expression in some cell strains (RH8/9/10), but to a lesser extent than HepG2 cells. The functional status of these RH cells was thought to resemble that of 10- to 17-day-old fetal rat hepatocytes. ➢Establishment of unique method to specifically immortalize ALB-expressing cells. Cultures were cotransfected with puromycin resistance gene under control of ALB enhancer/promoter. Only cells derived from hepatocytes were obtained after drug selection.	[24]
	Electroporation
	SV40 Tag
SVHepB4	Rat adult hepatocytes	-Displayed a hepatocyte-like morphology. -Maintained significant activity and inducibility of phase I and phase II enzymes. -Showed low activity of GGT, a hepatocyte dedifferentiation or transformation marker, whereas the hepatospecific enzyme TAT was expressed at levels similar to those in liver. -Possessed no anchorage-independent growth.	[178]
	Wild type simian virus strain LP
	SV40 Tag
Conditional immortalization
	Rat adult hepatocytes	-Immortalized cells cultured at permissive temperature (33°C) expressed low mRNA and protein levels of ALB, ASGPR and androsterone-UGT but high mRNA and protein levels of GST-Yp, compared to 24 hours primary rat hepatocyte cultures. - Immortalized cells cultured at non-permissive temperature (37°C): ■ Displayed morphologic characteristics of differentiated hepatocytes. ■ Expressed increased mRNA and protein levels of ALB, ASGPR, and androsterone-UGT. ■ Possessed markedly decreased GST-Yp expression. -Transplantation into SCID mice revealed no tumorigenic properties and cells retained hepatocellular morphology and G6P activity. ➢Expression of ASGR was found to be temperature-sensitive with higher expression at 37°C compared to 39°C. Consequently after SV40 Tag degradation, cells were maintained at 37°C instead of 39°C.	[118]
	Retroviral vector
	SV40 Tag
	Temperature-based regulation
	Rat adult hepatocytes	-Displayed morphology of primary hepatocytes. -Expressed ALB, G6P, DPP-IV, GGT mRNA levels similar to normal hepatocytes and ALB levels were stable for at least a month. -Secreted an amount of ALB equivalent to primary hepatocytes, however once transplanted in hepatectomized nonalbumin rats, immortalized hepatocytes did not generate significantly elevated ALB levels compared with primary hepatocytes. -Were not tumorigenic.	[129, 147]
	Human artificial mini chromosome
	SV40 Tag
	Recombinase- based control (FLP/FRT)
BQ1 BV1 WA1 WB6	Rat adult hepatocytes	-Displayed typical epithelial cell morphology. -Cultured at non-permissive temperature (37°C) expressed decreased levels of p53. -Although increased at non-permissive temperature, production of ALB and urea was still low compared to primary hepatocytes and reached undetectable levels with passage. -Possessed increased telomerase activity following immortalization but no anchorage-independent cell growth was visible. -Distribution of chromosomes was adapted during passaging. Significant increase of tetraploid along with passage (WA1/WB6/BQ1). For BV1 cells no significant change of chromosome count with passage, though several common chromosomal aberrations, namely, trisomy, monosomy and unknown marker chromosome noted only in higher passages. ➢It was demonstrated that conditionally immortalized hepatocytes become dedifferentiated by in vitro passage.	[102]
	Retroviral vector
	SV40 Tag
	Temperature-based regulation
C8-B	Rat adult hepatocytes	- Following Cre-recombinase treatment: ■ Regained morphological characteristic of differentiated hepatocytes. ■ Showed increased mRNA levels of ALB, HNF4 and UGT1 and newly detectable UGT2 and ASGPR mRNA. -Possessed no anchorage-independent colony formation or tumor production. ➢Ad-Cre infection was not 100% efficient and some cells did not undergo recombination.	[84]
	Retroviral vector
	SV40 Tag
	Recombinase- based control (Cre-LoxP)
H2.35	Mice adult hepatocytes	-Cultured at permissive temperature exhibit extremely low ALB mRNA levels. -Cultured at non-permissive temperature in serum-free medium and collagen substratum possessed dramatically increased ALB mRNA levels.	[120]
	Simian virus 40
	SV40 Tag
	Temperature-based regulation
L2A2	Rat adult hepatocytes	-Cultured at non-permissive temperature (37°C/39°C) regained the morphological characteristics of differentiated hepatocytes -Possessed increased albumin production at non-permissive temperature -Integrated normally into line cords and appeared indistinguishable from native liver parenchymal cells when transplanted into normal rat liver. -Once engrafted in the spleen of portacaval-shunted rats, displayed normal morphology, secreted bile, expressed ALB mRNA and offered protection from hyperammonemia-induced hepatic encephalopathy. -Were not tumorigenic.	[119, 179]
	Retroviral vector
	SV40 Tag
	Temperature-based regulation