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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: J Hepatol. 2014 Jun 6;61(4):925–943. doi: 10.1016/j.jhep.2014.05.046

Table 2. Overview of the available immortalization strategies.

HSV-TK, herpes simplex virus thymidine kinase; hTERT, human telomerase reverse transcriptase; n.a., not applicable; SV40 Tag, simian virus 40 large T antigen

Immortalizing genes
Rodent adult hepatocytes Human adult hepatocytes Human fetal hepatocytes
Viral oncogenes
  • -

    Allow the cells to overcome the proposed in vitro telomere-independent growth arrest.

  • -

    Immortalization of cells.
  • -

    Allow the cells to overcome the proposed in vitro telomere-independent growth arrest.

  • -

    Expansion of in vitro lifespan.
  • -

    Expansion of in vitro lifespan.
hTERT n.a
  • -

    Contradicting results available

  • -

    Does not allow the cells to overcome the proposed in vitro telomere-independent growth arrest.
  • -

    Immortalization of cells.
Viral oncogenes + hTERT n.a
  • -

    Immortalization of cells.
  • -

    Immortalization of cells.

  • -

    Use of oncogenes may be needed to overcome premature growth arrest which occurs when cells are cultivated under inappropriate culture conditions [77, 108].
Conditional immortalization
Advantage Disadvantage
Temperature-based regulation
  • -

    Cell growth can easily be manipulated by temperature shift.

  • -

    SV40 Tag not active at physiological temperature.
  • -

    The method is restricted to the temperature-sensitive SV40 Tag mutant.

  • -

    The immortalization gene is not removed from genome leading to potential risk for tumorigenesis when used in vivo.

  • -

    Temperature shift can induce changes in cellular properties and complicate interpretation of study outcome.
Recombinase-based regulation
  • -

    Excision of immortalizing gene upon Cre recombinase expression offers more possibilities for in vivo applications.
  • -

    Associated with an irreversible growth arrest.

  • -

    Proper reversion depends on efficient transfer of recombinase gene.

  • -

    Risk for chromosomal rearrangement by recombinase activity.
+ negative selection marker (HSV-TK) Cells that underwent improper recombination can efficiently be eliminated by ganciclovir exposure.
+ tamoxifen-mediated self-excision Elevates the need of a secondary virus-mediated transfer of the recombinase gene.
Transcriptional regulation
  • -

    Allows switching between the proliferating and growth arrest state.

  • -

    Expression of immortalizing gene can be controlled in vivo.
  • -

    Leaky transgene expression.
Gene transfer
Non-viral
Calcium phosphate precipitation
  • -

    High hepatocyte toxicity.

  • -

    Low gene transfer efficiencies.
Strontium phosphate transfection
  • -

    Low hepatocyte toxicity.

  • -

    Low gene transfer efficiencies.
Electroporation
  • -

    High hepatocyte toxicity.

  • -

    Low gene transfer efficiencies.
Lipid-mediated gene transfer
  • -

    When optimized, good gene transfer efficiencies can be obtained.

  • -

    Use of hepatocyte-specific ligands as transfection vehicle can lead to hepatocyte-specific transfections.
Viral
Retroviral
  • -

    Not able to transduce non-dividing cells.
Lentiviral
  • -

    Transduce both dividing and non-dividing cells.

  • -

    Transduction without affecting the differentiated phenotype.

  • -

    Improvement of lentiviral transduction efficiency by addition of growth factors and vitamin E to the culture medium.
Human artificial chromosomes
  • -

    Lower transfer efficiency than viral vectors.

  • -

    Mitotically stable episomal maintenance.

  • -

    Allows incorporation of large genes under control of their regulatory elements.