Cell immortalization, the process of bestowing cells the ability to proliferate indefinitely, is a cornerstone in biomedical research. It paves the way for long-term studies, fostering discoveries in cancer biology, drug development, and much more. Several methods have been developed to achieve cell immortalization, each with its unique advantages and applications. Here, we delve into some of these methods, highlighting their benefits and realms of application.

Telomerase Reverse Transcriptase (hTERT) Immortalization

Telomerase Reverse Transcriptase (hTERT) Immortalization is a molecular biology technique used to extend the replicative lifespan of human cells by introducing the human telomerase reverse transcriptase gene (hTERT) into them[1].

Here are some of the key advantages of hTERT immortalization in comparison to other approaches:

1. Natural-Like Telomere Maintenance:

hTERT immortalization extends cell lifespan by maintaining telomeres, which is a natural mechanism found in many immortal cell types. This approach mimics the mechanism employed by germ cells, stem cells, and some cancer cells to avoid telomere shortening and senescence. Other methods may rely on artificial mechanisms, such as viral oncogenes, that do not occur naturally.

2.No Requirement for Tumorigenic Genes:

Some methods of cell immortalization involve the introduction of genes with tumorigenic potential, such as viral oncogenes (e.g., SV40 Large T antigen) or mutant forms of tumor suppressor genes (e.g., p53). These genes can carry a higher risk of uncontrolled cell growth and may not be suitable for all research purposes. hTERT immortalization, in contrast, focuses specifically on telomere maintenance without introducing tumorigenic genes.

3.Long-Term Stability:

Immortalized cell lines generated using hTERT typically maintain stability over extended periods, providing a consistent and reliable resource for research. This is essential for long-term experiments and studies requiring a continuous supply of cells.

As we all know, Telomerase reverse transcriptase (hTERT) immortalization has a wide range of applications, here are a few key applications:

1.stem cell research:

Immortalization with hTERT enables the long-term culture and expansion of stem cells, including adult stem cells and induced pluripotent stem cells (iPSCs). This is essential for obtaining sufficient cell numbers for experimentation and therapeutic applications.

2. Drug Discovery and Development:

hTERT-immortalized cancer cell lines are essential tools for screening potential anticancer drugs. Researchers can assess the efficacy of new drug candidates and study their mechanisms of action, helping to identify promising therapies.Immortalized cells with controlled telomerase expression can be used for testing the effects of drugs on cells affected by telomere shortening. This is particularly relevant in drug discovery for age-related diseases or conditions influenced by telomere length.

3. Ideal for cells significantly affected by telomere length.

The primary purpose of hTERT immortalization is to extend the replicative lifespan of cells by preventing telomere shortening. Cells with critically short telomeres would otherwise undergo replicative senescence, leading to growth arrest. Immortalizing cells with hTERT allows researchers to extend the life of these cells without causing significant changes in their characteristics, making them more suitable for studying specific cell types or diseases.

4. Understanding Telomere Biology:

The use of hTERT immortalization helps researchers explore telomere biology in the context of stem cells, shedding light on how telomere maintenance impacts stem cell function and pluripotency.

Viral Oncogene Immortalization

Viral oncogene immortalization is a technique used to extend the replicative lifespan of cells by introducing specific viral genes known as oncogenes. Several viral oncogenes have been commonly used for immortalization, including:

SV40 Large T Antigen:

Derived from the Simian Virus 40 (SV40), Large T Antigen disrupts the functions of tumor suppressor proteins like p53 and pRb, leading to uncontrolled cell division.

HPV E6 and E7:

Human Papillomavirus (HPV) E6 and E7 oncoproteins target and inactivate p53 and pRb, respectively.


Epstein-Barr Virus (EBV) Latent Membrane Protein 1 can activate various signaling pathways involved in cell proliferation and survival.

Here are some of the advantages of viral oncogene immortalization:

1. Well-established:

Viral oncogene immortalization is often a highly efficient process, rapidly extending the replicative lifespan of target cells.

2. Predictable Immortalization:

The introduction of specific viral oncogenes leads to predictable outcomes in terms of cell immortalization. Researchers can select oncogenes with well-defined mechanisms of action, ensuring consistency in the immortalization process.

3. Reliable Replicative Lifespan Extension:

Viral oncogene immortalization typically results in a stable and substantial extension of the replicative lifespan of target cells. This stability allows for long-term experiments and the generation of reliable and consistent cell lines.

Next, we will introduce some applications of Viral Oncogene Immortalization:

1. Establishment of Stable Cell Lines:

Viral oncogene immortalization allows researchers to create stable and continuously proliferating cell lines from primary or finite-lifespan cells. This is particularly useful for maintaining a consistent source of cells for experiments over extended periods.

2. Viral oncogene immortalization can be used for general research purposes where maintaining the original cell characteristics is not a primary concern.

This method is particularly useful when the emphasis is on obtaining long-lasting and rapidly proliferating cell lines for various experiments, and precise preservation of the original cell characteristics is not essential. For research aimed at understanding fundamental cellular processes such as cell cycle regulation, apoptosis, or signal transduction pathways, maintaining original cell characteristics may not be necessary.

3. Studies on viral oncogenesis:

Modeling Viral-Induced Transformation: Viral oncogene immortalization allows researchers to establish in vitro models of viral-induced cellular transformation. This involves introducing specific viral oncogenes into target cells to mimic the cellular changes caused by oncogenic viruses. These models are essential for understanding the early events in viral oncogenesis.


SV40 Large T Antigen Immortalization

SV40 Large T Antigen (SV40 LT Ag) immortalization is a widely used technique in cell biology and molecular biology to extend the replicative lifespan of mammalian cells[2]. SV40 LT Ag is derived from the Simian Virus 40 (SV40) and is known for its ability to disrupt the functions of tumor suppressor proteins, such as p53 and pRb (retinoblastoma protein). By inactivating these proteins, SV40 LT Ag enables cells to bypass senescence and continue dividing, making it a powerful tool for creating immortalized cell lines.



1. High efficiency: SV40 LT Ag immortalization is known for its high efficiency. It can rapidly extend the replicative lifespan of target cells, allowing for the creation of immortalized cell lines in a relatively short time.

2. SV40 LT Ag immortalization can be applied to a variety of cell types, including primary cells and cell lines derived from different tissues.

3. Induces telomerase activity, aiding in telomere maintenance. SV40 LT Ag-induced activation of telomerase results in increased telomerase activity. This leads to the extension of telomeres at the ends of chromosomes during cell division. Telomere extension helps prevent telomere shortening, which is a hallmark of cellular aging and senescence. As a result, SV40 LT Ag-immortalized cells can divide indefinitely without experiencing telomere-related replicative senescence.


1. SV40 LT Ag-immortalized cell lines are used to study various aspects of cancer biology, including the molecular mechanisms underlying tumorigenesis, cancer progression, and drug resistance.

2. Molecular Biology Studies:SV40 LT Ag-immortalized cells serve as model systems for investigating cellular processes, such as cell cycle regulation, DNA replication, and DNA repair.

HPV E6/E7 Immortalization

HPV E6/E7 immortalization is a technique used to extend the replicative lifespan of cells by introducing two key viral genes, E6 and E7, derived from high-risk human papillomaviruses (HPV)[3]. HPV E6 and E7 oncoproteins have the ability to modulate cellular pathways, inhibit the functions of tumor suppressor proteins, and promote uncontrolled cell growth, ultimately leading to cell immortalization.


1. Effective in immortalizing epithelial cells: HPV E6/E7 immortalization is highly effective at immortalizing epithelial cells. Epithelial cells are the primary target of high-risk human papillomaviruses (HPV), and HPV E6 and E7 oncoproteins have evolved to interact specifically with cellular proteins in these cells, leading to their transformation and immortalization.

2. Specificity for Epithelial Cells: HPV E6/E7 oncoproteins are specifically tailored to interact with cellular proteins in epithelial cells. This makes them highly effective for immortalizing epithelial cell types, which are the primary targets of HPV infections.

3. Bypasses p53 and Rb tumor suppressor pathways, promoting cell cycle progression: HPV E6/E7-immortalized cells are used for molecular biology research to investigate cell cycle regulation, DNA repair, and various cellular processes affected by the disruption of p53 and pRb functions.


1. HPV E6/E7-immortalized cell lines are valuable for studying the molecular mechanisms of HPV-associated cancers, such as cervical cancer.

2. Researchers can use HPV E6/E7-immortalized cells to create in vitro disease models that mimic the cellular changes associated with HPV infection and the development of HPV-related diseases.HPV E6/E7-immortalized epithelial cell lines are widely used in research related to HPV infections, including the study of viral replication, pathogenesis, oncogenesis, and the development of therapeutic strategies and vaccines.

Chemical or Physical Immortalization

Chemical immortalization involves the use of chemical compounds or agents to achieve the immortalization of cells. These compounds typically act on specific cellular pathways or components to prevent senescence or cell death.

Physical immortalization involves physical processes or alterations to the cells to extend their replicative lifespan. This can include mechanical, electrical, or other physical interventions.


Advantages of Chemical Immortalization:

1. Chemical immortalization allows researchers to have precise control over the process. They can choose specific compounds or agents that target particular cellular pathways or processes, tailoring the approach to their research goals.

2. Simple and cost-effective.

3. In some cases, chemical interventions for immortalization can be reversible. Researchers can remove the chemicals or agents to revert the cells back to their original state, providing flexibility in experimental design.

4. Chemical methods, such as telomerase introduction, typically carry a lower risk of introducing genomic instability compared to some physical methods or viral oncogene-based approaches.

Advantages of Physical Immortalization:

1. Physical immortalization methods, such as viral oncogene introduction, are known for their high efficiency in extending the replicative lifespan of cells. They can rapidly generate immortalized cell lines.

2. Immortalized cell lines created through physical methods are often stable over extended periods. They maintain their characteristics and proliferative capacity, providing a consistent and long-lasting source of cells.

3. Does not require genetic engineering.


1. Chemical immortalization is used to create stable, long-lasting cell lines that can be propagated in culture for extended periods. Immortalized cell lines generated through chemical methods are employed in high-throughput drug screening assays.

2. Physical immortalization methods, particularly those involving viral oncogenes, are widely used in cancer research to create cell models that mimic aspects of cancer cell behavior. They are valuable for studying oncogenic processes, drug responses, and potential therapeutic targets.

Spontaneous Immortalization

Spontaneous immortalization refers to a natural process by which some cells, under certain conditions, become immortalized without deliberate external intervention. It occurs when cells continue to divide and proliferate beyond their normal replicative limit without undergoing senescence or cell death. Spontaneous immortalization typically involves genetic alterations that enable cells to escape normal regulatory mechanisms that limit cell division.


1. Natural process without exogenous intervention.Spontaneously immortalized cell lines can be readily obtained from naturally occurring biological samples, such as tissues or primary cultures.

2. Spontaneously immortalized cell lines are less likely to have undergone extensive genetic manipulation compared to some artificially immortalized lines. This can be an advantage when studying the natural biology of cells.

Useful for studying the mechanisms of cellular aging and immortalization.



1. Spontaneously immortalized cells can be studied alongside their parental cells to understand the factors and genetic changes responsible for bypassing cellular senescence.

2. Precisely because spontaneous immortalization is a natural process in which cells become immortal without deliberate external intervention, the natural mechanisms of cellular immortalization can be well understood.

Each of these methods unveils a pathway to transcend the boundaries of cellular aging, opening doors to an array of research possibilities. The choice of method largely depends on the cell type, the research goal, and the importance of maintaining the original cellular characteristics. Through these diverse methods, the scientific community continues to unravel the mysteries of cellular processes, propelling the biomedical research field towards new horizons.

KMD Bioscience has extensive research experience in cell immortalisation. We have already successfully constructed the immortalized cell lines derived from human, mouse and rat. Based on the dedicated scientific teams and advanced experimental platforms, KMD Bioscience has developed an efficient virus transfection technology. We offer a comprehensive range of reagents and engineered cell lines to meet your research needs.

Visit https://www.kmdbioscience.com/pages/cell-immortalization-platform.html to have a detailed understanding.



Yik MY, Azlan A, Rajasegaran Y, Rosli A, Yusoff NM, Moses EJ. Mechanism of Human Telomerase Reverse Transcriptase (hTERT) Regulation and Clinical Impacts in Leukemia. Genes (Basel). 2021 Jul 30;12(8):1188. doi: 10.3390/genes12081188. PMID: 34440361; PMCID: PMC8392866.

Bryan TM, Reddel RR. SV40-induced immortalization of human cells. Crit Rev Oncog. 1994;5(4):331-57. doi: 10.1615/critrevoncog.v5.i4.10. PMID: 7711112.

Hawley-Nelson P, Vousden KH, Hubbert NL, Lowy DR, Schiller JT. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 1989 Dec 1;8(12):3905-10. doi: 10.1002/j.1460-2075.1989.tb08570.x. PMID: 2555178; PMCID: PMC402081.