Cell Immortality in the Lab: How Scientists Make Cells Live Forever — and Why It Matters

An in-depth expansion of Chalak et al. (2024), Life 14, 417, with verified supporting sources.

Why anyone would want a cell that never dies

Most cells removed from a human or animal body and placed in a petri dish behave a little like a clock that has been wound only so many times: they divide, they divide again, and then they stop. The reasons are practical, not philosophical. Fresh tissue has to be obtained from a donor — sometimes by an invasive biopsy. Each batch comes with a different age, sex, hormonal milieu, and genetic background, so experiments done one month may not reproduce the next. There are ethical questions about harvesting from people and animals, paperwork, costs, and the simple fact that mature cells from sources like vascular tissue are notoriously hard to isolate. As the 2024 review by Chalak and colleagues puts it, "the repeated procurement of cells requires much skill and experience," and the variability between donors can lead to "errors and changes in the interpretation and results of research."

The workaround is to engineer cells that bypass senescence entirely. These so-called immortalized cell lines divide indefinitely, can be grown at industrial scale, and spare researchers the chore of going back to a donor every few months. They have powered everything from the polio vaccine to cultured-meat research. But immortality in a dish comes in several flavors, some far safer than others, and the techniques used to achieve it carry their own baggage — genomic instability, contamination, and a 70-year-old ethical shadow cast by the most famous cell line of all time.

The Hayflick limit: a counting mechanism built into chromosomes

The story begins with Leonard Hayflick. Working at the Wistar Institute in Philadelphia in 1961, Hayflick and Paul Moorhead showed that a normal human fetal cell population will divide between roughly 40 and 60 times in culture before entering a non-dividing state called senescence. Chalak et al. cite the rounder figure of "approximately 60 times population doubling." This ceiling — later named the "Hayflick limit" by the Nobel laureate Macfarlane Burnet in his 1974 book Intrinsic Mutagenesis — overturned a long-held dogma. Before Hayflick, the surgeon Alexis Carrel had claimed that chicken heart fibroblasts could be kept dividing forever, and that he had maintained such a culture for 34 years. Other scientists could never reproduce Carrel's result, and the consensus today is that fresh embryonic cells were probably being added to his cultures by accident, replenishing the population while making it look "immortal."

The molecular basis of the Hayflick limit is the gradual shortening of telomeres, the protective caps of repetitive DNA that sit at the ends of linear chromosomes. In all vertebrates the repeat unit is the six-base sequence TTAGGG. In humans, telomere length ranges from about 0.5 to 15 kilobases, depending on tissue type, donor age, and replication history, with chromosome 17p typically carrying shorter telomeres than other chromosome ends. Each time a normal somatic cell divides, the unidirectional DNA replication machinery cannot quite copy the very tip of the strand — the so-called "end replication problem" first articulated by James Watson — and telomeres erode by roughly 50 to 200 base pairs per division. When telomeres become critically short, the cell reads them as double-stranded DNA breaks, mounts a DNA-damage response, and either commits apoptosis or enters the growth-arrested state known as the M1 (mortality stage 1) phase.

The crucial insight came in the 1970s from Alexei Olovnikov, who proposed that the repeated telomeric sequences act as a buffer, and that the length of that buffer determines how many divisions a cell can afford. In 1986, Cooke and Smith provided the first direct evidence linking telomeres to aging when they showed that the telomeres covering sex chromosomes in sperm cells are far longer than in adult somatic cells — implying that adult cells must lose telomeric DNA over time. Subsequent work confirmed telomere shortening in dividing fibroblasts and in aging blood and colon cells, but not in germline cells, which maintain their telomeres via the enzyme telomerase.

Real-world numbers sharpen the picture. The University of Utah's Genetic Science Learning Center reports that in white blood cells, telomere length drops from about 8,000 base pairs at birth to roughly 3,000 in adults and as low as 1,500 in the elderly. A 2013 review in Epidemiologic Reviews estimated the loss at about 50 base pairs per cell division. Across species, telomere lengths vary widely: human telomeres typically run 10–15 kb, rat and some mouse strains carry telomeres up to 150 kb, yeast telomeres are around 300 bp, and the ciliate Tetrahymena — where telomerase was first discovered — has only 20–70 hexameric repeats.

Telomerase: the enzyme that resets the clock

If telomere erosion is what kills normal cells in culture, then a cell that could rebuild its telomeres would, in principle, live forever. That cell exists. Telomerase is a large ribonucleoprotein complex that adds TTAGGG repeats back onto chromosome ends. It was first detected in 1985 as an activity that extends telomeric sequences in Tetrahymena, and its protein components were finally identified and characterized in 1997. The enzyme has two essential subunits: a catalytic reverse transcriptase called hTERT, encoded by the TERT gene, and an RNA template called hTERC (also written hTR), encoded by the TERC gene. The hTERT gene spans roughly 35 kb and contains 16 exons and 15 introns; it produces a 1,132-amino-acid polypeptide that matures into a 130-kilodalton functional protein with four key domains — the N-terminal regulatory domain, the RNA-binding domain, the reverse transcriptase domain, and the C-terminal dimerization domain.

Telomerase also depends on accessory proteins (DKC1, NOP10, NHP2, GAR1) and on a six-protein shelterin complex (TRF1, TRF2, RAP1, POT1, TINF2, TPP1) that protects the very ends of chromosomes from being mistaken for damage. The enzyme is highly active in proliferating cells, hematopoietic stem cells, germline cells, and rapidly renewing tissues — but is repressed in most adult somatic cells, largely through tight transcriptional control of hTERT. This repression is what makes telomere shortening a tumor-suppressive mechanism in the first place: it limits how many times a potentially dangerous somatic cell can copy itself.

HeLa: the accidental origin of cell immortality

The first human cell line to be grown indefinitely in vitro did not come from a clever telomerase experiment — it came from a tumor. In February 1951, a 31-year-old African American woman named Henrietta Lacks was admitted to Johns Hopkins Hospital in Baltimore with abnormal vaginal bleeding. She was diagnosed with cervical cancer. During her treatment, doctors took samples of her tumor — without her knowledge or consent, as was customary at the time — and passed them to George Otto Gey, head of tissue culture research at Johns Hopkins. Unlike most cells, which died within days in culture, Lacks's cells grew explosively and never stopped. They were dubbed "HeLa," using the first two letters of her first and last name. Henrietta Lacks died on October 4, 1951, but her cells are still dividing in laboratories around the world today.

HeLa cells are immortal because they express an active version of telomerase that keeps rebuilding their telomeres during every cell cycle, allowing them to evade the Hayflick limit. The line has been used in more than 74,000 research studies and has contributed to a remarkable string of breakthroughs: Jonas Salk's polio vaccine, the discovery that human papillomavirus (HPV) causes cervical cancer, the development of HPV vaccines, the understanding of telomerase itself, gene mapping, in vitro fertilization, AIDS treatments, COVID-19 research, and three Nobel-Prize-winning bodies of work. They have been commercialized by dozens of biotechnology companies.

But the HeLa story is also one of the foundational ethical scandals of modern medicine. Henrietta Lacks's family was unaware that her cells were being grown and sold until the 1970s, when researchers began contacting them for genetic testing. For decades the family received no compensation even as companies profited from products derived from her tissue. In August 2023, the Lacks estate reached a confidential settlement with Thermo Fisher Scientific. In February 2026, the estate settled with Novartis Pharmaceuticals; in March 2026, it resolved claims against Viatris. Litigation against Ultragenyx Pharmaceutical remained active as of early 2026. The case directly shaped the U.S. "Common Rule," introduced in 1991, which set ethical guidelines for informed consent in human research — and it remains a touchstone for debates about tissue ownership, racial justice in medicine, and the commercialization of biological materials.

HeLa also created a quieter, more technical headache that still plagues labs today. Because HeLa cells grow so aggressively, they can quietly take over other cell cultures — a single contaminated pipette tip is enough. Walter Nelson-Rees exposed the scale of the problem in the early 1970s, showing that a significant fraction of cell lines distributed by major cell banks were actually HeLa in disguise. The review notes that a 2011 study of 122 head and neck cancer cell lines found that 30% were misidentified, and broader analyses of tissue-culture collections in the U.S., Europe, and Asia suggest that at least 15% of cell lines in circulation are misidentified or contaminated. Mycoplasma contamination adds another layer of invisible trouble, silently altering gene expression and behavior in cultures that look perfectly healthy under a microscope.

How scientists actually immortalize a cell today

The 2024 review organizes the toolbox into six families: viral oncoproteins, overexpression of specific immortalizing genes, exploitation of cancer-derived cells, combination strategies, chemical and physical carcinogens, and CRISPR-based gene editing. Each makes different tradeoffs between efficacy, safety, and how "normal" the resulting cell line remains.

1. Viral oncoproteins: hijacking the cell cycle

Viruses depend on forcing host cells into S phase so they can replicate their own genomes, and they have evolved elegant ways to do it. The most widely used viral tool is the SV40 Large T antigen, which binds and inactivates the tumor suppressors p53 and the retinoblastoma protein (pRb) — the two proteins that enforce the M1 senescence barrier. SV40 T antigen is 708 amino acids long and also targets p107, p130, the CBP/p300 and p400 adaptors, and (via the small T antigen) the phosphatase pp2A. It has been used to immortalize human proximal tubular epithelial cells (which keep their differentiation properties) and microglial cells, which Garcia-Mesa and colleagues used to study HIV latency in the central nervous system. But SV40 T can cause problems: pre-adipocytes immortalized with it differentiate abnormally, because T antigen blocks the p300/CBP transcription factor required for adipogenesis.

Human papillomavirus takes a similar two-pronged approach with its E6 and E7 oncoproteins. High-risk strains (HPV-16/18/31/8) cause malignant lesions; low-risk strains (HPV-6/11) cause warts. In immortalization, E6 activates telomerase and accelerates proteasomal degradation of p53, while E7 prevents pRb from binding the E2F transcription factor, freeing cells to enter S phase. Trakarnsanga and colleagues used HPV-16 oncoproteins to immortalize adult erythroblasts, producing stable cell lines that can manufacture red blood cells — a route toward lab-grown blood for transfusion. HTLV-1 and HTLV-2, the retroviruses behind adult T-cell leukemia, encode Tax1 and Tax2; Tax2 is more active in driving T-cell proliferation despite HTLV-2 being clinically less pathogenic. Adenovirus contributes the 12S E1A oncoprotein, which can establish primary epithelial cells as continuous lines while preserving expression of keratin and vimentin. Epstein–Barr virus (EBV) immortalizes B lymphocytes by converting them into lymphoblastoid cell lines (LCLs); EBNA-1, EBNA-2, and LMP are each required, with EBNA-2 controlling viral and cellular RNA accumulation and EBNA-1 maintaining viral DNA replication. LCLs are widely used in pharmacogenomic studies of how genetic variation affects drug response.

The Chalak review is explicit about the cost: viral oncoproteins disable the very tumor-suppressor pathways that keep cells from turning malignant, leading to genomic instability, polyploidy, and altered chromosome numbers. p53 is inactivated in roughly half of all human malignant cancers, underscoring why disabling it deliberately is not "safe and risk-free."

2. Overexpression of cellular genes: a gentler approach

Several non-viral genes can push cells past senescence, though many of them are themselves oncogenes. Members of the HOX family — HOX11/TLX1, Hox-2.4, HOXa9 — immortalize specific hematopoietic progenitors (erythroid, megakaryocytic, monocytic, myelocytic, and multipotent cells). Lhx2 immortalizes multipotent hematopoietic stem/progenitor cells that retain the ability to produce erythroid, myeloid, and lymphoid lineages after grafting into mice. The myc family (c-Myc, N-Myc, L-Myc, B-Myc) is the most studied; c-Myc is expressed in proliferating cells and is itself linked to p53 status. Overexpressing c-Myc drives DNA damage and genomic instability while bypassing p53's pro-apoptotic function; NF-κB malfunction is a necessary condition for Myc-induced carcinogenesis. De Filippis used a c-Myc T58A mutant delivered by retroviral vector to immortalize neural stem cells, while Li used L-Myc to generate a perpetual NSC population. c-Myc-immortalized human prostate epithelial cells (HPrECs) maintain normal p53 responses and lack gross karyotypic abnormalities, but they lose the Rb/p16INK4a checkpoint — a real weakness. CDK4 combined with hTERT has been used to immortalize human bronchial epithelial cells, and CDK4 with cyclin D1 plus telomerase activity has been used for myogenic cells derived from healthy and diseased muscle.

3. hTERT: the gold-standard "safe" method

For most researchers who want a cell line that behaves like the original primary cell but can be passaged forever, overexpressing hTERT alone is the preferred route. Unlike viral oncoproteins, hTERT is not an oncogene — it does not transform cells. hTERT-immortalized cells typically retain normal cell-cycle control with active p53 and pRb checkpoints, normal contact inhibition, a requirement for growth factors, and a stable karyotype even at high passage numbers. They do not progress to malignancy in culture, and in many studies they have been differentiated into tissue-specific cell types that form structures resembling those in the body.

The review catalogues an impressive range of hTERT-immortalized primary cells: fibroblasts, choroidal melanocytes, endothelial cells, dermal keratinocytes, mammary epithelial cells, osteoblasts, pancreatic cells, human vocal fold epithelial cells (which kept dividing stably for more than 8 months while preserving ion- and protein-transport functions), primary canine corneal epithelial cells, yak rumen epithelial cells, RPE-1 retinal pigment epithelial cells (versatile for gene editing via ERT2-Cre-ERT2), HUVECs (the first confirmation of antitumor immunity in hTERT-immortalized endothelial cells), bone-marrow mesenchymal stem cells (capable of forming bone, supporting stroma, and adipocytes when transplanted into immune-deficient mice), and human fetal hepatocytes (when combined with SV40 T and E7). A 2024 review in the International Journal of Molecular Sciences reinforced this consensus, arguing that hTERT expression level, telomere length, and senescence-associated β-galactosidase staining are all somewhat controversial readouts, and proposing a standard assessment protocol that checks proliferation rate, morphology, phenotype, karyotype stability, telomerase activity, cell-specific markers, and tumorigenicity.

4. Cancer cells: when nature did the immortalization for you

Some cells become immortal without any help from a lab — they are tumor cells, taken from a biopsy and propagated. The first immortalized fibroblast line was derived from mouse fibroblasts in the 1940s; HeLa is the most famous human example. A particularly interesting subfield involves cancer-associated fibroblasts (CAFs), the activated stromal cells that surround tumors. CAFs promote tumor growth, angiogenesis, metastasis, and chemotherapy resistance, and primary CAFs isolated from human carcinomas remain active for long periods in culture. Epigenetic events — particularly DNA methylation — shape CAF identity: TGF-β1 from cancer cells suppresses miR-200, which increases DNMT3B expression; miR-141 inhibition lifts TCF12, which drives CXCL12 secretion by CAFs, which in turn upregulates c-Myc and cyclin D1 in nearby breast cancer cells. The review cautions, however, that deliberately steering primary cells toward malignancy via epigenetic manipulation cannot be considered safe or reliable.

5. Combination strategies: when one tool isn't enough

Single methods often fail for stubborn cell types. The review notes that suppressing p53 with small interfering RNA can delay senescence but cannot alone immortalize human ovarian surface epithelial cells or mesenchymal stem cells; introducing hTERT alone is also insufficient. Combining the two, however, works. WI-38 embryonic lung fibroblasts — a workhorse for aging research — have been made non-dividing via combined overexpression of T antigen, hTERT, and H-ras. The general principle: pair a telomerase reactivation with one or more cell-cycle checkpoint disruptors, and tune the cocktail to the cell type.

6. Chemical carcinogens and radiation: the old-school route

Cells can also be pushed past senescence by exposure to genotoxic and non-genotoxic carcinogens or to ionizing radiation. Genotoxic agents like N-methyl-N-nitrosourea (MNU) and benzo(a)pyrene damage DNA directly and have been shown to immortalize Syrian hamster dermal cells by inactivating p53 and p16. Methyl sulfate, a potent clastogen, works similarly in mammalian SDH cells and Chinese hamster cells. X-rays, neutrons, and gamma rays produce immortal clones in SHD cells, although immortalization of human mammary cells by ionizing radiation is rare. The carcinogenic potency of basic aliphatic alkylating agents is directly linked to their ability to modify the O6 atom of guanine. Two familiar drugs have unexpected immortalization-relevant effects: acetaminophen has been reported to activate telomerase (while also inhibiting CDK4/CDK2 and imposing a G1 checkpoint), and aspirin at therapeutic concentrations prevents endothelial senescence by increasing nitric oxide synthesis and reducing oxidative stress, while also lowering the level of asymmetric dimethylarginine (an endogenous NO synthase inhibitor) and reducing DDAH activity. These observations sit at the boundary between immortalization research and pharmacology, and they remain biologically interesting rather than practically deployed.

7. CRISPR/Cas9: the new precision tool

The newest entry in the immortalization toolbox is CRISPR/Cas9. By inducing targeted double-strand breaks and using the cell's homology-directed repair machinery, CRISPR allows immortalizing constructs to be knocked into precise "safe harbor" loci rather than being inserted semi-randomly by a virus — a meaningful improvement in safety and reproducibility. The review highlights two examples: mouse bone-marrow mesenchymal stem cells reversibly immortalized by targeting SV40T to the Rosa26 locus, generating the so-called imBMSCs; and primary common marmoset skin fibroblasts immortalized by Cas9-mediated targeting of p53 or the CDKN2A locus. The marmoset is a valuable non-human primate model, and CDKN2A-deficient marmoset cells — like their mouse counterparts — express wild-type p53 and respond normally to genotoxic stressors like adriamycin and etoposide. CRISPR is also being used to make immortalized cell lines more amenable to downstream editing, stable transgene integration, and reporter construction.

What immortalized cells are actually used for

The applications of immortalization read like a tour of modern biomedicine. They underpin vaccine development, drug metabolism and toxicity testing, antibody production, gene-function studies, artificial tissue (synthetic skin substitutes like StrataGraft, used in burn care), and the manufacture of therapeutic proteins. In neuroscience, the NTera2 (NT2) line has been used in clinical trials for brain-injury grafting; the CTX0E03 human neural stem cell line rescues behavioral and pathological deficits in a Huntington's disease rodent model. Encapsulated cell technology, which delivers genetically modified therapeutic proteins from macro-capsules, has been limited by the lack of a compatible allogeneic human cell line — exactly the gap immortalization can fill. iPSC-derived megakaryocytes immortalized via TET-on-regulated c-MYC can produce platelets for clinical trials. EBV-transformed lymphoblastoid cell lines are workhorses for pharmacogenomic studies of how genetic diversity shapes drug safety and efficacy. And the new field of cultured meat depends on immortalized cells to provide safe, large-scale, animal-biopsy-free production of muscle and fat tissue — the review identifies this as one of the most important emerging frontiers for immortalization technology.

The persistent problems

Immortalized cell lines are not magic. The review and the wider literature flag four recurring issues:

  1. Genetic and phenotypic drift. Continuous passage can shift genotypic and phenotypic features, producing genetic drift and heterogeneity within cultures. Cell lines therefore may not mirror all attributes of primary cells in the long term, and may yield different results in different labs.
  2. Cross-contamination and misidentification. As noted above, the 2011 study of 122 head and neck cancer lines found 30% misidentified; broader estimates put the rate at ≥15% across all lines. A faster-growing contaminant can quietly dominate a culture. HeLa contamination, first exposed by Walter Nelson-Rees in the 1970s, remains the textbook case.
  3. Mycoplasma contamination. Often undetected for long periods, mycoplasma substantially alters gene expression and cellular behavior, undermining conclusions drawn from "clean" looking cultures.
  4. Safety of the immortalization method itself. Disabling p53 and pRb, or inserting viral oncogenes, can produce karyotypic instability and, in some cases, tumorigenic potential. The review explicitly recommends not using viral oncoprotein strategies where a non-oncogenic route such as hTERT overexpression would suffice, and that key findings obtained in immortalized lines should be confirmed in primary cultures whenever possible.

Bottom line: if you want to act on this today, here is what you can do

If you are starting a project that needs dividing human or animal cells for more than a few months, the practical playbook is fairly clear:

  • Pick hTERT over viral oncoproteins whenever possible. hTERT-only immortalization preserves p53/pRb checkpoints, contact inhibition, growth-factor dependence, and a near-normal karyotype. Reserve SV40 T, HPV E6/E7, EBV, or adenovirus E1A for cell types where hTERT alone fails, and combine them only as needed (e.g., hTERT + p53 suppression for ovarian surface epithelial cells).
  • Combine methods only when a single method is insufficient. Pair telomerase reactivation with a single checkpoint disruptor, and document the resulting karyotype against the parent primary cells.
  • Use CRISPR/Cas9 for targeted insertion. Knock immortalizing constructs into a defined safe-harbor locus (such as Rosa26) rather than relying on random viral integration; this improves safety and reproducibility, especially for downstream clinical or cell-therapy applications.
  • Authenticate and test every line. STR-profile or SNP-authenticate new lines on arrival and after expansion; test for mycoplasma on a regular schedule; and confirm that the karyotype, morphology, proliferation rate, and tissue-specific markers still match the parent primary cells. A 2024 protocol from Shitova and colleagues offers a useful checklist.
  • Validate key findings in primary cells. Treat the immortalized line as a workhorse, not a substitute. Critical mechanistic claims should be confirmed in primary cultures or in vivo models.
  • If your work touches HeLa or any tissue-derived product, mind the ethics. Know the history of the cells you use, the consent status of the original donor (or lack thereof), and the ongoing legal landscape — the 2023 Thermo Fisher settlement and the 2026 Novartis and Viatris settlements with the Lacks estate show that this is now an active area of litigation, not just a historical footnote.
  • If you are in cell agriculture or biomanufacturing, plan for scale. Immortalized lines are particularly attractive for cultured meat and biologic production because they eliminate the need for repeated animal biopsies, but they must meet food-safety criteria and exhibit the expected taste, texture, and nutritional profile.

In short: telomerase-first, CRISPR-targeted, meticulously authenticated, and ethically aware. The technology to make cells live forever is mature; the harder problems now are quality control, reproducibility, and consent.

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