hTERT-immortalized primary cells are cells that have been genetically modified to express the human telomerase reverse transcriptase (hTERT) gene, enabling them to bypass senescence and divide indefinitely. Unlike other immortalization techniques, hTERT overexpression maintains many of the original characteristics of primary cells, allowing for long-term culture without significant genetic changes.
Mechanism of hTERT Immortalization
Telomerase and Cellular Senescence
In most somatic cells, the enzyme telomerase is not active, leading to the progressive shortening of telomeres with each cell division. When telomeres become critically short, the cell enters senescence (growth arrest) or undergoes apoptosis.
hTERT is the catalytic subunit of the enzyme telomerase, which elongates telomeres, preventing their shortening and allowing cells to continue dividing indefinitely.
hTERT Overexpression
By introducing the hTERT gene into primary cells, researchers can reactivate telomerase, preventing telomere shortening and enabling cells to proliferate beyond their normal lifespan while preserving their normal physiological functions.
hTERT Immortalization Protocol
Here’s a generalized protocol for immortalizing primary cells with hTERT.
Materials
Primary cells (e.g., fibroblasts, epithelial cells)
Lentiviral or retroviral vector containing the hTERT gene
Lentiviral or retroviral packaging plasmids (if producing your own virus)
Transfection reagent (e.g., Lipofectamine 2000)
HEK 293T cells (for viral packaging, if producing your own virus)
Growth medium (e.g., DMEM or RPMI-1640)
Antibiotic for selection (e.g., puromycin or neomycin)
Polybrene (for enhanced transduction efficiency)
Incubator set to 37°C with 5% CO₂
Day 1: Virus Production (if not using pre-made virus)
HEK 293T Transfection
Seed HEK 293T cells (or another packaging cell line) at approximately 70-80% confluency in a 10 cm dish.
Transfect the cells with the hTERT-expressing vector and the packaging plasmids using a transfection reagent like Lipofectamine 2000 or PEI.
After 6-8 hours, replace the medium with fresh complete DMEM containing 10% FBS.
Harvest Lentiviral/Retroviral Supernatant
Collect the virus-containing supernatant from the transfected cells 48-72 hours after transfection.
Filter the supernatant through a 0.45 µm filter to remove cell debris.
Concentrate the viral particles using ultracentrifugation (optional) or use them directly for transduction.
Day 3: Transduction of Primary Cells
Prepare Primary Cells
Seed your primary cells (e.g., fibroblasts, epithelial cells) in a 6-well or 12-well plate at 50-70% confluency.
Lentiviral/Retroviral Transduction
Add 5-10 µg/mL of polybrene to the culture medium to enhance transduction efficiency.
Add the viral supernatant containing hTERT to the cells at an appropriate multiplicity of infection (MOI). A higher MOI will increase the likelihood of successful transduction.
Incubate the cells with the viral supernatant for 24 hours at 37°C.
Medium Replacement
After 24 hours, replace the viral supernatant with fresh complete medium and continue to culture the cells for 48-72 hours to allow for stable expression of hTERT.
Day 6: Selection of Transduced Cells (Optional)
Antibiotic Selection
If the hTERT-expressing vector contains an antibiotic resistance gene (e.g., puromycin or neomycin), begin selection by adding the appropriate antibiotic to the culture medium.
Use puromycin (1-2 µg/mL) or neomycin/G418 (400-800 µg/mL), depending on the vector.
Maintain selection for 3-5 days until non-transduced cells are eliminated and only antibiotic-resistant cells remain.
Expand Selected Cells
Once selection is complete, allow the surviving hTERT-expressing cells to expand in fresh medium. Change the medium every 2-3 days.
Day 10+: Validation and Expansion
Validate hTERT Expression
Confirm the expression of hTERT in the immortalized cells by using quantitative PCR (qPCR) or Western blotting.
Assess telomerase activity using the TRAP assay (Telomeric Repeat Amplification Protocol), which measures the activity of telomerase.
Proliferation and Senescence Assays
Perform proliferation assays (e.g., cell counting, MTT) to confirm that the hTERT-immortalized cells have an extended lifespan compared to non-immortalized cells.
Stain the cells for β-galactosidase (a marker of senescence) to ensure that the immortalized cells are not senescent.
Cryopreservation
Once the immortalized cells are proliferating stably, freeze them in freezing medium (e.g., 10% DMSO in FBS) for long-term storage.
Advantages of hTERT Immortalization
Preservation of Cell Properties: Unlike immortalization methods involving viral oncoproteins (e.g., SV40 or HPV), hTERT immortalization usually preserves the cells’ normal physiology, differentiation potential, and genomic stability.
Extended Proliferative Capacity: hTERT allows cells to proliferate indefinitely, making it possible to conduct long-term studies that would not be feasible with primary cells alone.
Low Risk of Tumorigenicity: Since hTERT immortalization does not directly interfere with tumor suppressor pathways (like p53 or pRb), the cells are less likely to become tumorigenic compared to other methods.
Examples of hTERT-Immortalized Cell Lines
BJ-hTERT (Human Foreskin Fibroblasts)
Derived from human foreskin fibroblasts, these cells are commonly used for studying fibroblast biology, skin aging, and wound healing.
The cells maintain typical fibroblast morphology and characteristics while being able to proliferate indefinitely.
N/TERT-1 and N/TERT-2 (hTERT-Immortalized Keratinocytes)
Human epidermal keratinocytes immortalized with hTERT, widely used to study skin biology, wound healing, and skin disorders.
These cells retain many of the characteristics of normal keratinocytes, including their ability to differentiate in response to calcium and other signals.
HUVEC-hTERT (Human Umbilical Vein Endothelial Cells)
These endothelial cells, immortalized with hTERT, are often used for studies of angiogenesis, vascular biology, and cardiovascular diseases.
The cells retain their ability to form tube-like structures in vitro, a characteristic of endothelial cells.
RPE1-hTERT (Retinal Pigment Epithelial Cells)
Retinal pigment epithelial cells immortalized with hTERT are used for research in ophthalmology, particularly for studying retinal degeneration and age-related macular degeneration (AMD).
MSC-hTERT (Mesenchymal Stem Cells)
Mesenchymal stem cells from bone marrow or adipose tissue are immortalized with hTERT to study stem cell biology, differentiation, and regenerative medicine applications.
Applications of hTERT-Immortalized Cells
Cancer Research
hTERT-immortalized cells are often used to study the role of telomerase in cancer development and to investigate how cancer cells avoid senescence.
Aging Research
These cells are essential for studying cellular aging, telomere biology, and the molecular mechanisms that drive cellular senescence.
Regenerative Medicine and Tissue Engineering
hTERT-immortalized cells serve as models for developing tissue-engineered constructs and studying stem cell differentiation and tissue repair processes.
Drug Discovery and Toxicology
Immortalized primary cells are valuable tools for high-throughput drug screening and evaluating the cytotoxicity or efficacy of new therapeutic compounds.
Basic Cell Biology
These cell lines provide a renewable, physiologically relevant model system for studying fundamental cellular processes, such as cell cycle regulation, DNA repair, and apoptosis.
hTERT immortalization is a powerful method to generate cell lines with extended proliferative capacity while maintaining many of the original characteristics of the primary cells. These cell lines are essential for a wide variety of research applications, including aging studies, cancer biology, and regenerative medicine.