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. Author manuscript; available in PMC: 2015 Sep 23.
Published in final edited form as: Methods Mol Biol. 2015;1250:53–76. doi: 10.1007/978-1-4939-2074-7_4

Table 1. Overview of immortalization strategies, hepatic functionality and in vitro applications of human adult and fetal hepatic cell lines.

(A1AT, α1-antitrypsin; AFP, α-fetoprotein; AhR, aryl hydrocarbon receptor; ALB, albumin; A2M, α2-macroglobulin; APO, apolipoprotein; Arnt, AhR nuclear translocator; ASGP(R), asialoglycoprotein (receptor); BCRP, breast cancer resistance protein; BSEP, bile salt export pump; CAR, constitutive androstane receptor; C/EBP, Ccaat-enhancer-binding protein; ChREBP, carbohydrate-responsive element-binding protein; CK, cytokeratin; CLDN, claudin; CYP, cytochrome P450; EH, epoxide hydrolase; EMT, epithelial-mesenchymal transition; EPCAM, epithelial cell adhesion molecule; FXR, farnesoid X receptor; GGT, γ-glutamyltranspeptidase; G6P, glucose-6-phosphate; GPX, gluthatione peroxidase; GS, glutamine synthetase; GST, gluthatione S-transferase; HBCF, human blood coagulation factor; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HGFR, hepatocyte growth factor receptor; HNF, hepatocyte nuclear factor; HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase; IL, interleukin; IFN, 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; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; Rb, retinoblastoma; RIPK4, receptor-interacting serine-threonine kinase 4; SOD, superoxide dismutase; SREBP, sterol regulatory element-binding protein; SV40 Tag, simian virus 40 large T antigen; TGF, transforming growth factor; TF, transferrin; (bil-)UGT, (bilirubin-)uridinediphosphate-glucuronosyltransferase).

Adult hepatic cell line
Cell line Immortalization strategy Hepatic functionality of immortalized cells In vitro applications Reference
Fa2N-4 Transfection
  • -
    Possess, 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.
  • -

    Are incapable of metabolizing compounds due to low basal levels of drug-metabolizing enzymes.

  • -

    Exhibit, 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 lose the ability to induce.

 Routine screening system for PXR-mediated CYP3A4 induction. (18,92,97)
SV40 Tag
HepLi5 Retroviral vector
  • -

    Express HBCF-X, GS, GST, ALB and CYP450 mRNA.

  • -

    Retain ALB secretion and urea production, though at low levels compared to primary hepatocytes.

  • -

    Display CYP1A2 activity.

  • -

    Possess significantly enhanced cellular functions after large-scale culture in roller bottles.

 (21)
SV40 Tag
HepLL Lipid mediated gene transfer (lipofectamine reagent)
  • -

    Display morphologic characteristics of liver parenchymal cells.

  • -

    Express HNF4, HBCF-X, GST-Π and ALB mRNA as well as ALB and CYP2E1 protein but no ASGP mRNA.

  • -

    Stain positive for human hepatocyte special antigen but negative for AFP.

  • -

    Secrete ALB and urea at levels not significantly different from primary cultured human hepatocytes.

  • -

    Synthesize glycogen.

 Testing of new drug carriers for anti-HBV drug delivery. (22, 98)
SV40 Tag
HepZ Lipid mediated gene transfer (lipofectamine reagent)
  • -

    When grown in bioreactor, cells are able to secrete ALB and A2M and possess inducible CYP450 activity.

 (47)
Antisense constructions for Rb and p53 under control of ALB promoter + Cotransfection of E2F transcription factors and cyclin D1
HHE6E7T-1/2 Small hepatocytes
  • -

    Display epithelial-like morphology.

  • -

    Retain characteristics of differentiated hepatocytes, though functions such as ALB secretion as well as mRNA expression levels of ALB, HNF4 and A1AT decrease gradually as the passages progress. CK18 mRNA levels are detected throughout the culture period and no AFP expression is observed.

  • -

    Are positive for vimentin staining.

 (16, 99)
Lentiviral and retroviral vectors
HPV16 E6/E7 + hTERT
HHL(-5/-7/-16) Retroviral vector
  • -

    Contain markers of hepatocyte and biliary phenotype (CK7/8/18/19).

  • -

    Express CYP450 protein at levels comparable to Huh-7 and HepG2 cells.

  • -

    Produce ALB, though at lower levels than Huh-7 and HepG2 cells.

  • -

    Stain negative for AFP and do not display elevated nuclear expression of p53 protein.

  • -

    Possess active gap junctions.

  • -

    Respond to INF-α stimulation by upregulation of major histocompatibility complex I and II

  • -

    Exhibit, in contrast to the Huh-7 and HepG2 cells, increased capacity to bind recombinant hepatitis C virus-like particles.

 Used as a cell model to investigate:
  • -

    Ability of CD8+ T-cells to recognise and kill hepatocytes under cytokine stimulation.

  • -

    Role of sirtuin 4 in energy homeostasis.

  • -

    Effect of THCV on insulin signalling in insulin-resistant human hepatoctyes.

  • -

    Gene expression and antigen presentation after adeno-associated viral transduction and effect of proteasome inhibition or capsid mutation.

 (15, 89-91, 100-102)
HPV16 E6/E7
IHH-A5 Lipid mediated gene transfer (lipofectin reagent)
  • -

    Are morphologically and functionally more similar to hepatoma cell lines than primary hepatocytes in culture.

  • -

    Secrete 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 results in increased fibrinogen secretion and decreased ALB production, demonstrating a proper acute-phase response.

  • -

    Produce detectable amounts of APO-A1.

  • -

    Exhibit bile-canalicular structures that, in some cases, accumulated the organic anion glutathione-methylfluorescein. Cell cultures are partly polarized and express the efflux transporters, MDR1 and MRP1, on the membranes of apical vacuoles or on the lateral membranes of adjacent, proliferating cells, respectively.

  • -

    Do not maintain active Na+ -dependent bile salt uptake.

  • -

    Display similar lipoprotein metabolism as HepG2 cells.

 Used a cell model to investigate:
  • -

    Molecular mechanisms underlying FXR regulation of ChREBP transcriptional activity in human hepatocytes.

  • -

    Effect of antipsychotic drugs on SREBP transcription factor pathways which control.

  • -

    Role of c-fos expression on hepatocyte cell motility and cell cycle regulation.

  • -

    Effect of cadmium on nonmalignant human hepatocytes.

 (23, 85, 86, 103-105)
SV40 Tag
PH5CH Lipid mediated gene transfer (lipofectin reagent)
  • -

    Display epithelial appearance.

  • -

    Express human CK and ALB protein.

 Used a cell model to investigate:
  • -

    HCV infection, replication and tropism.

  • -

    HCC-selective cytotoxicity of HBF-0079.

  • -

    MicroRNA expression in TGF-β-induced hepatocyte EMT.

  • -

    Effect of hepatitis B virus proteins on signalling mediated by members of the Toll-like/interleukin 1 superfamily.

  • -

    Effect of HBV polymerase on IFN production in human hepatocytes.

 (25, 74, 75, 81, 106-113)
SV40 Tag
THLE Retroviral vector
  • -

    Display epithelial morphology.

  • -

    Secrete ALB and express CK18, TF, A1AT, A2M, GST-Π and very low levels of GGT at early passages. CK19 expression can only be determined at later passages. Cells are uniformly negative for AFP and factor VIII. The appearance of CK19 and decreased ALB secretion at later passages demonstrate that cells undergo dedifferentiation in culture.

  • -

    Retain mRNA expression of phase II enzymes such as EH, catalase, GPX, SOD and GSTs at levels comparable to human liver, with GST-Π and a mRNA as the dominant form in THLE cells or human liver, respectively.

  • -

    Maintain NADPH CYP reductase expression at a lower steady-state mRNA level than in human liver.

  • -

    Are able to metabolize three carcinogens, which suggests the presence and activity of CYP1A2/1A1, CYP2E1 and CYP3A4. However CYP1A2, CYP2E1, CYP3A4, CYP2A3 and CYP2D6 mRNA are not detected. The steady-state mRNA levels of CYP1A1 increase after exposure to Aroclor 1254 or B[α] P.



  • ➢

    Overexpression of specific CYP450 gene led to the development of THLE-CYP sublines.

 THLE and THLE-CYP cells can be used to study cellular toxicity of compounds.
Used a cell model to investigate:
  • -

    HCC-selective cytotoxicity of HBF-0079.

  • -

    Hepatoprotective and chemopreventive properties of phytochemicals.

 (25, 93, 94, 111, 114-124)
SV40 Tag
TPH1 Strontium phosphate precipitation
  • -

    Exhibit altered cell morphology resembling low-differentiated epithelial cells.

  • -

    Express no A1AT or AFP mRNA.

  • -

    Secrete ALB.

  • -

    Possess G6P activity.

  • -

    Reactivate telomerase immediately after senescence.

 Used a cell model to investigate HCV infection and replication.

Induces apoptosis of activated hepatic stellate cells.		 (45, 77, 78, 125-129)
HCV core gene
Fetal and neonatal hepatic cell lines
Cell line Immortalization strategy Hepatic functionality of immortalized cells In vitro applications Ref
FH-TERT Retroviral vector
  • -

    Express CYP450 mRNA and maintain, in contrast to passaged fetal hepatocytes, liver-enriched differentiation markers, especially C/EBPα and HNF4 as well as elevated levels of HGFR.

  • -

    Possess glycogen storage and G6P activity, in a pattern similar to primary fetal hepatocytes.

  • -

    Produce urea and retain level of ALB synthesis equivalent to HepG2 cells.



  • ➢

    Culture conditions used in these studies were designed at supporting cell proliferation; conditions have not been optimized for inducing differentiated hepatocellular functions.

 Used as a stroma to induce human embryonic stem cellsdifferentiation into hematopoietic cells.

Used as a cell model to investigate:
  • -

    Role of RIPK4 as novel tumor suppressor in human hepatocarcinogenesis.

  • -

    Permissive role of β-catenin signalling in the initial phase of hepatocarcinogenesis.

 (34, 83, 84, 130)
hTERT
Hc3716-hTERT Retroviral vector
  • -

    Maintain normal mammalian cell morphology.

  • -

    Exhibit protein expression of ALB, CK8 and CK18, but not AFP. ALB levels are higher than in control, passaged Hc3716 cells.

  • -

    Possess inducible CYP3A4/7 mRNA levels.

  • -

    Exhibit wild-type p53 responsiveness.

 Used as in vitro model for predicting the side-effects of telomere-targeting drugs. (39)
hTERT
NeHepLxHT Retroviral vector
  • -

    Display characteristic morphology of primary fetal liver cells.

  • -

    Maintain epithelial characteristics as evidenced by immunostaining for epithelial cell markers, the cytokeratins.

  • -

    Possess 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 indicate the progenitor nature of the cells.

 Used as a cell model to investigate:
  • -

    Role of FAT10 in promoting malignant cell transformation.

  • -

    Molecular mechanism responsible for miR-224 overexpression in HCC.

  • -

    Role of HCV RNA-dependent RNA polymerase in promoting liver inflammation and injury.

 (14, 80, 131, 132)
hTERT
OUMS-29 Lipid mediated gene transfer (lipofectin reagent)
  • -

    Display epithelial morphology.

  • -

    Maintain gene expression of ALB, ASGPR, bil-UGT, GS, GST-Π, HBCF-X, AhR and Amt.

  • -

    Secrete ALB, AFP, TF, A1AT and APO A-1.

  • -

    Possess inducible CYP1A1/2 mRNA levels and activity.



  • ➢

    Overexpression of HNF4α2 led to development of OUMS-29/H-11 cell line with increased liver-specific gene expression, such as A1AT, apolipoproteins, HBCF-X and HNF1α.

 Used as a cell model to investigate:
  • -

    Role T-cell factor 4 isoforms in promoting hepatic tumorigenicity.

  • -

    Effect of bile acid species on hepatocyte apoptosis induced by PPARgamma ligands.

  • -

    Effect of hydrogen peroxide on hepatic pigment epithelium-derived factor levels.

  • -

    Both morphologic and functional alterations in the mitochondria oftroglitazone-treated hepatocytes.

 (17, 49, 62, 133-136)
SV40 Tag