Cell immortalization is defined as the process by which cells acquire the capacity to proliferate indefinitely, overcoming the normal replicative senescence caused by telomere shortening and other intrinsic checkpoint mechanisms. While naturally occurring in tumorigenic cells, immortalization can also be artificially induced in vitro through genetic, viral, or enzymatic methods. Immortalized cell lines play a crucial role in biological research, biotechnology, and regenerative medicine. The timeline below summarizes the principal milestones in the development of cell immortalization technologies and their applications.
Early Foundations (1900s–1950s)
- 1907 – Ross G. Harrison develops the first method for in vitro cell culture using frog neural tissue, laying the groundwork for tissue culture methodologies.
- 1943 – Earle et al. establish the first spontaneously immortalized mouse fibroblast cell line (L929), used widely for cytotoxicity testing.
- 1951 – George Gey establishes the HeLa cell line from Henrietta Lacks’ cervical carcinoma. HeLa cells become the first human immortal cell line and a cornerstone of experimental cell biology.
- 1965 – Leonard Hayflick and Paul Moorhead define the Hayflick limit, demonstrating that normal human fibroblasts undergo a finite number of divisions before entering senescence.
1970s–1980s: Viral Oncogenes and Telomerase Discovery
- 1973 – Graham et al. generate HEK293 cells by integrating adenovirus 5 DNA into human embryonic kidney cells. These cells become a key tool in molecular biology and protein expression.
- 1970s–1980s – Viral oncogenes such as SV40 large T antigen, Adenovirus E1A/E1B, and Epstein–Barr virus (EBV) are used to immortalize primary human and rodent cells by inactivating tumor suppressor pathways (e.g., p53 and Rb).
- 1984 – Carol Greider and Elizabeth Blackburn discover telomerase, a ribonucleoprotein enzyme that maintains telomere length and is active in immortal and cancer cells.
- 1989 – The human telomerase reverse transcriptase (hTERT) gene is cloned and identified as the catalytic subunit of telomerase.
1990s: Genetic and Conditional Immortalization
- 1994 – Development of temperature-sensitive SV40 large T antigen mutants (e.g., tsA58) enables conditionally immortalized cell lines that can switch between proliferative and growth-arrested states.
- 1998 – Bodnar et al. demonstrate that hTERT overexpression alone is sufficient to extend the lifespan and immortalize normal human somatic cells without malignant transformation, providing a safer alternative to viral oncogenes.
2000s: Standardization and Broader Applications
- 2001–2004 – Protocols for hTERT-mediated immortalization of various human primary cell types (e.g., epithelial, endothelial, mesenchymal stem cells) are published and widely adopted.
- Hybridoma technology, originally developed in the 1970s by Köhler and Milstein, becomes a mainstay for generating immortalized antibody-producing B cell lines through fusion with myeloma cells.
- 2000s – Techniques for immortalizing post-mitotic or lineage-specific cells (e.g., myoblasts, osteoblasts, pancreatic β-cells) using combinations of hTERT, CDK4, and oncogenes (e.g., c-Myc) are introduced.
2010s: Precision Gene Editing and Reversible Immortalization
- 2012 – The introduction of CRISPR–Cas9 genome editing enables precise targeting of genes involved in cell cycle control and senescence, allowing the generation of genetically defined immortalized cell lines.
- Mid-2010s – Inducible immortalization systems (e.g., doxycycline-inducible hTERT, Cre-loxP-regulated SV40 T antigen, tamoxifen-inducible c-MycER) are developed, allowing temporal control of the immortalization process and enhancing safety in translational research.
2020s: Immortalization of Rare and iPSC-Derived Cell Types
- 2020–2023 – Advances in stem cell biology and organoid technology lead to the successful immortalization of iPSC-derived cell types (e.g., neurons, hepatocytes, pancreatic cells), enabling disease modeling and high-throughput drug screening.
- Recent studies focus on spontaneous immortalization in non-human models (e.g., avian and fish cells) and uncover novel mechanisms of replicative escape, potentially informing human applications.
- New technologies also explore transgene-free immortalization using small molecules, epigenetic modifiers, and long non-coding RNAs.
