Cell immortalization offers a significant solution for extending the proliferative capacity of primary cells in culture, enabling long-term and reproducible research applications. Primary cells are directly isolated from fresh tissue and closely mimic in vivo conditions, making them ideal for various experiments. However, they eventually stop dividing, called replicative senescence. This biological limitation restricts their use for a long-term research project. Researchers often need to harvest fresh cells repeatedly, leading to batch-to-batch variability and reduced experimental reproducibility. Immortalization addresses these issues by modifying critical cellular mechanisms of proliferation, such as altering telomere regulation or suppressing senescence signaling pathways, allowing cells to bypass growth arrest and divide indefinitely. The following are several tools for immortalizing cells in culture conditions.
Introducing TERT (telomerase reverse transcriptase) is a common method for immortalizing human cells that alters telomere regulation. Telomerase is a ribonucleoprotein enzyme that extends the DNA sequence of telomeres at the ends of chromosomes. By maintaining telomere length, telomerase enables cells to bypass replicative senescence and continue dividing indefinitely. While telomerase is typically inactive in most somatic cells, it is highly active in tumor cells. Taking advantage of this mechanism, TERT overexpression is widely used to achieve stable cell immortalization in research and biotechnology applications.
Another approach to cell immortalization involves the inactivation of tumor suppressor genes, such as p53 and Rb. These genes normally function to prevent uncontrolled cell proliferation. When they are repressed or inactivated, cells can bypass senescence and continue dividing.
One common method to achieve this is by introducing viral oncogenes, which are derived from viruses known to induce cancer. These oncogenes can inactivate tumor suppressor proteins and promote continuous cell growth. The most widely used viral oncogene is SV40 large T antigen, which inactivates both p53 and Rb. Other examples include:
EBV genes (EBNA1/EBNA2) - commonly used for B cell immortalization
HPV16 E6/E7 - effective in immortalizing keratinocytes
E1A gene from human adenovirus type 5 - used for epithelial cell immortalization, e.g. HEK293 is a human embryonic kidney cell immortalized by E1A of adenovirus 5.
c-Myc - a cellular oncogene that can drive proliferation and extend lifespan in some cell types.
Additionally, siRNAs targeting p53 or Rb can also be employed to transiently suppress these genes and support immortalization.
Cell immortalization service follows a standard and effective process to generate immortalized cell lines. The process starts with isolating primary cells from tissue samples, carefully preserving their original characteristics. Once isolated, we confirm their identity and the purity of the cells using immunostaining with a specific surface marker. Then, an immortalizing gene (e.g. SV40 T antigen) is introduced into the isolated cells with a resistance gene using lentiviral vectors. Antibiotic selection is then applied to enrich for successfully transduced cells. The surviving cells are allowed to grow and expand, forming an immortalized cell line. Immunostaining is finally performed again to check that the cells still show the expected markers and characteristics. This step-by-step process helps us create high-quality immortalized cell lines that are ready for your research or other applications.
Please contact us with a brief description of your project or specific need (the cells you want to immortalize and the method you may be interested in). One of our experts will get in touch with you soon to discuss your requirement and support you with a customized strategy for free.
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