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Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges
Open accessAbstract
Cells are very important to researchers due to their use in various biological studies in in vitro and in vivo settings. This importance stems from the short lifespan of most cells under laboratory conditions, which can pose significant challenges, such as the difficulties associated with extraction from the source tissue, ethical concerns about separating cells from human or animal models, limited cell passage ability, and variation in results due to differences in the source of the obtained cells, among other issues. In general, cells in laboratory conditions can divide into a limited number, known as the Hayflick limit, due to telomere erosion at the end of each cellular cycle. Given this problem, researchers require cell lines that do not enter the senescence phase after a limited number of divisions. This can allow for more stable studies over time, prevent the laborious work associated with cell separation and repeated cultivation, and save time and money in research projects. The aim of this review is to summarize the function and effect of immortalization techniques, various methods, their advantages and disadvantages, and ultimately the application of immortalization and cell line production in various research fields.
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TL;DR
This review summarizes in vitro techniques for cell immortalization, including viral oncoproteins, oncogene overexpression, hTERT, chemical/radiation exposure, and CRISPR/Cas9, and discusses applications and challenges. It concludes that immortalized cell lines are valuable but carry risks such as genomic instability, phenotypic drift, and contamination.
Key findings
- Cells normally face the Hayflick limit after approximately 60 population doublings due to telomere shortening; immortalized lines bypass senescence, with HeLa being the first successful human cell immortalization and having active telomerase.
- Major immortalization methods include viral oncoproteins (SV40 large/small T antigen, HPV E6/E7, HTLV Tax1/Tax2, adenovirus 12S E1A, EBV), overexpression of genes such as HOX/TLX1, Lhx2, c-Myc, CDK4, and hTERT, cancer-associated fibroblasts, combined methods, chemical/radiation exposure, and CRISPR/Cas9 gene editing.
- hTERT overexpression is presented as a safer approach because hTERT is not an oncogene; hTERT-immortalized cells can retain normal cell cycle control, active p53 and pRb checkpoints, contact inhibition, growth factor dependence, and stable karyotypes.
- Applications include vaccine development, drug testing, antibody production, gene function studies, artificial tissues, therapeutic proteins, regenerative medicine, and cultured meat; challenges include cell line misidentification, with one 2011 study finding 37 of 122 head and neck cancer cell lines (30%) misidentified and analyses suggesting at least 15% of cell lines are misidentified or contaminated.
Methods
- Design: narrative review of in vitro cell immortalization techniques, their advantages and disadvantages, and applications.
- Data or sample: no experimental sample or primary data are reported; the paper synthesizes published literature and cited studies.
- Main analysis: descriptive/thematic synthesis of immortalization mechanisms, including viral oncoproteins, gene overexpression, cancer cells, combined methods, chemical/radiation exposure, and CRISPR/Cas9.
Limitations
- The paper states that cell lines may not fully mirror primary cells because genetic manipulation can alter phenotype, inherent functions, and responses to stimuli; repeated cultivation can cause genetic drift and heterogeneity, and contamination by other cell lines or mycoplasma can persist undetected and alter gene expression and cellular behavior.
- The paper states that viral oncoprotein methods inactivate p53 and Rb, can lead to genomic instability, polyploidy, and altered chromosome numbers, and are not considered safe or risk-free; p53 loss is observed in about half of human malignant cancers.
- Evident from the text: as a narrative review, no systematic search strategy, inclusion/exclusion criteria, or quantitative synthesis is described, and no primary experimental validation is reported.
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In the paper
“The immortalized NTera2 (NT2) cell line has undergone a thorough examination for its potential in brain grafting. Notably, these cells have been safely employed in clinical trials focused on treating brain injuries [36,37].”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 2. The Application of Immortalized Cell Lines
What the sources say
- Not checked — no full text of the sourcecites [36] Richardson, 2007, Ectopic telomerase expression inhibits neuronal differentiation of NT2 neural progenitor cells, Neurosci. Lett. source
- Not checked — no full text of the sourcecites [37] Hara, 2008, Neural progenitor NT2N cell lines from teratocarcinoma for transplantation therapy in stroke, Prog. Neurobiol. source
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Types 1 and 2 encode for the Tax1 and Tax2 proteins, respectively, which are necessary for the infection of human T-cells by the associated viruses.”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 4. Immortality and a Review of Its Techniques > 4.1. Viral Genes Can Control the Cell Cycle
What the sources say
- Check pendingcites [79] Sieburg, 2004, Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis, J. Virol. source
plainstates a cause- Asserted: passes — The authors put it forward as true.
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In the paper
“Conversely, pre-adipocytes showed aberrant differentiation after immortalization with the SV40 T antigen. In fact, the ability of the SV40 T antigen to block the transcription factor p300/cAMP-response element-binding protein (CBP), which is essential for adipocyte differentiation, inhibits pre-adipocyte differentiation [71].”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 4. Immortality and a Review of Its Techniques > 4.1. Viral Genes Can Control the Cell Cycle
What the sources say
- Not checked — no full text of the sourcecites [71] Darimont, 2003, Immortalization of human preadipocytes, Biochimie source
plainstates a cause- Asserted: passes — The authors put it forward as true.
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- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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In the paper
“For example, they have used immortalized cell lines as model systems for studying neuronal development and performance recovery in neurological disease models, such as Huntington’s disease [28,29].”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 2. The Application of Immortalized Cell Lines
What the sources say
- Not checked — no full text of the sourcecites [28] Geller, 1991, Applications of immortalized cells in basic and clinical neurology, J. Cell Biochem. source
- Check pendingcites [29] Yoon, 2020, Implantation of the clinical-grade human neural stem cell line, CTX0E03, rescues the behavioral and pathological deficits in the quinolinic acid-lesioned rodent model of Huntington’s disease, Stem Cells source
plain- Asserted: passes — The authors put it forward as true.
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- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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In the paper
“It has been shown that EBNA-2 is genetically essential for B cell immortalization by EBV.”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 4. Immortality and a Review of Its Techniques > 4.1. Viral Genes Can Control the Cell Cycle
What the sources say
- Not checked — no full text of the sourcecites [87] Middleton, 1991, Immortalizing genes of Epstein-Barr virus, Adv. Virus Res. source
boostedstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
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Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 2. The Application of Immortalized Cell Lines
“non-immortalized human lung epithelial cells created by hTERT overexpression without the use of viral oncogenes have been used to investigate various aspects of lung cancer”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > References
“34. Sato M. Shay J.W. Minna J.D. Immortalized normal human lung epithelial cell models for studying lung cancer biology”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 1. Introduction
“In the late 1950s, George Otto Gey, head of tissue culture research at Johns Hopkins, initiated the process of immortalization and established a non-aging cell line called HeLa, derived from a rare adenocarcinoma found in the cervix of a young woman named Henrietta Lacks.”
§ Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges > 1. Introduction
“Major discoveries have been made using this cell line, including the development of the polio vaccine in 1953 [25]”
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Metadata
- Authors
- Chalak, M., Hesaraki, M., Mirbahari, S. N., Yeganeh, M., Abdi, S., Rajabi, S., & Hemmatzadeh, F.
- Affiliations
- University of Science and Culture (IR)
- Royan Institute (IR)
- University of Tehran (IR)
- Adelaide University (AU)
- Corresponding author
- Rajabi, Sarah
- Hemmatzadeh, Farhid
- Venue
- Life
- Publisher
- MDPI AG
- Published
- 2024-03-21
- Volume
- 14
- Issue
- 3
- Pages
- 417
- ISSN
- 2075-1729 (ISSN-L)
- PMC
- PMC10971253
- OpenAlex
- W4393039470
- Cited by
- 66
- References
- 174
- Citations by year
- 2026: 28 · 2025: 29 · 2024: 9
- FWCI
- 2.71
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Life (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- University of Adelaide
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Keywords
- cell immortalization, cellular senescence, Hayflick limit, telomere shortening, cell lines, in vitro cell culture, subculture
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Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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