Cell immortalization refers to the process of modifying normal cells to allow them to proliferate indefinitely. Under natural circumstances, most cells have a limited ability to divide due to a phenomenon known as replicative senescence, which occurs as a result of telomere shortening and other cellular stress factors. Immortalization overcomes this limitation by enabling cells to bypass senescence and avoid programmed cell death (apoptosis), allowing them to grow continuously.

Immortalized cells are widely used in research because they provide a consistent, long-term source of cells for experiments, such as studying cellular functions, drug screening, cancer biology, and genetic studies.

Mechanisms of Cell Immortalization

There are several methods to immortalize cells, typically targeting pathways that control senescence and apoptosis. These methods involve altering genes or introducing factors that enable the cells to divide indefinitely.

Telomerase Activation

Telomeres are repetitive nucleotide sequences at the ends of chromosomes that protect them from damage. With each cell division, telomeres shorten, eventually leading to replicative senescence. This mechanism limits the number of divisions a cell can undergo, a phenomenon known as the Hayflick limit.

Telomerase is an enzyme that elongates telomeres, allowing cells to bypass senescence. In most somatic cells, telomerase is inactive, but it is highly active in certain cell types (e.g., germ cells, stem cells, and cancer cells).

Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, is commonly introduced into cells to reactivate telomerase and maintain telomere length, which extends the replicative lifespan of the cells. This method is often used to immortalize human fibroblasts and epithelial cells.

Viral Oncogenes (SV40 Large T Antigen)

One of the most common methods of immortalizing cells is by introducing viral oncogenes, such as SV40 large T antigen, derived from Simian virus 40 (SV40). This viral protein disrupts key tumor suppressor pathways, particularly those regulated by the p53 and Rb (retinoblastoma) proteins.

p53: A tumor suppressor that regulates cell cycle arrest and apoptosis in response to DNA damage or stress. SV40 large T antigen binds and inactivates p53, preventing apoptosis and allowing cells to continue dividing.

Rb (Retinoblastoma protein): Rb regulates the G1-S transition in the cell cycle. By binding to and inactivating Rb, SV40 large T antigen allows cells to bypass cell cycle checkpoints, leading to continuous proliferation.

SV40 large T antigen is widely used to immortalize a variety of cell types, including fibroblasts, epithelial cells, and neurons.

Human Papillomavirus (HPV) Oncoproteins (E6 and E7)

Human papillomavirus (HPV), particularly HPV16 and HPV18, contains two key oncogenes, E6 and E7, which can immortalize cells by interfering with the p53 and Rb pathways, similar to SV40 large T antigen.

E6: Promotes the degradation of p53, preventing cell cycle arrest and apoptosis.

E7: Binds and inactivates Rb, allowing cells to bypass the G1 checkpoint and proliferate.

HPV E6 and E7 are commonly used to immortalize human epithelial cells, such as keratinocytes and cervical cells.

hTERT Overexpression

As mentioned earlier, the human telomerase reverse transcriptase (hTERT) gene can be introduced into cells to maintain telomere length, preventing replicative senescence. This is a particularly clean method for immortalization because it does not affect the normal cell cycle control pathways (unlike SV40 T antigen or HPV E6/E7).

hTERT immortalization is widely used for human fibroblasts, epithelial cells, and endothelial cells, and it is preferred when studying cellular behavior without disrupting tumor suppressor pathways.

Bypass of the CDK Inhibitors

Cyclin-dependent kinase inhibitors (CDKIs), such as p16^INK4a, regulate the cell cycle by inhibiting cyclin-dependent kinases (CDKs) that control the progression from G1 to S phase.

In some immortalization methods, particularly in mouse cells, knocking down or silencing p16^INK4a (or its human equivalent p21^CIP1) can prevent cells from entering senescence and allow them to continue dividing.

Applications of Immortalized Cells

Basic Research

Immortalized cell lines provide a consistent and reproducible cell source for studying cellular functions, signaling pathways, and gene expression.

Examples of immortalized cell lines include HeLa cells (derived from cervical cancer), HEK293 cells (human embryonic kidney cells), and 3T3 fibroblasts (mouse fibroblasts).

Cancer Research

Since many cancer cells acquire immortality through similar mechanisms (e.g., reactivation of telomerase or p53 inactivation), immortalized cell lines serve as models for studying tumor biology, cancer progression, and the effects of anti-cancer drugs.

Drug Screening and Development

Immortalized cells are widely used in high-throughput screening assays to test the efficacy and toxicity of new drug candidates.

They provide a reproducible and scalable platform for screening large compound libraries in pharmaceutical research.

Gene Editing and Functional Studies

Immortalized cells are used in CRISPR-Cas9 and other gene-editing techniques to study the roles of specific genes in cellular processes such as proliferation, differentiation, and apoptosis.

Biomanufacturing

Immortalized cells can be engineered to produce therapeutic proteins, antibodies, or vaccines. Cell lines like Chinese hamster ovary (CHO) cells are widely used in the biotechnology industry for producing biopharmaceuticals.

Vaccine Production

Immortalized cell lines are commonly used in vaccine production, as they can grow rapidly and provide a reliable platform for propagating viruses or producing viral antigens.

Vero cells (derived from African green monkey kidney cells) are an example of an immortalized cell line used for vaccine production.

 Examples of Commonly Used Immortalized Cell Lines

HeLa Cells

Derived from cervical cancer, HeLa cells are one of the first and most widely used human cell lines in research. They were immortalized naturally by the HPV E6 and E7 oncoproteins.

HEK293 Cells

Human embryonic kidney cells immortalized with adenovirus E1A/E1B oncogenes. Widely used for gene expression, protein production, and drug screening.

NIH 3T3 Cells

Mouse embryonic fibroblasts that were spontaneously immortalized through serial passaging. Commonly used in molecular biology and cancer research.

CHO Cells

Chinese hamster ovary cells are used extensively in biomanufacturing for producing recombinant proteins, monoclonal antibodies, and vaccines.

293T Cells

A derivative of HEK293 cells, modified with SV40 large T antigen for enhanced gene expression. Frequently used for transfection and viral production.

Vero Cells

Derived from the kidney of an African green monkey, Vero cells are commonly used for virology research and vaccine production.

Challenges and Considerations

Genetic Stability

Immortalized cell lines can undergo genetic changes over time, leading to variability in behavior. This can be problematic for reproducibility across experiments.

Loss of Normal Cellular Phenotypes

While immortalization allows cells to proliferate indefinitely, it can also alter normal cell cycle control, differentiation capacity, or other phenotypic characteristics. Researchers need to validate that key functions of interest are preserved after immortalization.

Risk of Transformation

Some immortalization methods, particularly those using viral oncogenes (e.g., SV40 large T antigen or HPV E6/E7), can increase the risk of cell transformation and tumorigenicity. This should be considered when choosing the appropriate immortalization strategy.

Conclusion

Cell immortalization is a valuable technique for generating cell lines that can proliferate indefinitely, providing an unlimited source of cells for research, drug development, and biomanufacturing. Various methods, including telomerase activation, viral oncogene expression, and CDK inhibitor bypass, are used depending on the cell type and research goals. While immortalized cell lines offer many advantages, they must be carefully characterized to ensure they retain relevant biological properties for specific applications.