A cell immortalization protocol is a detailed set of instructions used to make primary cells proliferate indefinitely, bypassing their natural limit on division (senescence). The method used to immortalize cells can vary based on the specific technique (e.g., hTERT overexpression, SV40 Large T antigen, HPV E6/E7, etc.) and the type of cells being immortalized. Below is a general protocol for one of the most common methods: immortalization using human telomerase reverse transcriptase (hTERT).
Protocol for hTERT-Mediated Cell Immortalization
Materials
Primary cells (e.g., fibroblasts, epithelial cells)
Lentiviral vector containing hTERT (commercially available or self-constructed)
Lentiviral packaging plasmids (for lentivirus production)
Lipofectamine or other transfection reagent
HEK 293T cells (for producing lentiviral particles)
Polybrene (to enhance transduction efficiency)
Puromycin or another selection antibiotic (if using a selectable marker)
Dulbecco’s Modified Eagle Medium (DMEM) or another appropriate growth medium for primary cells
Fetal bovine serum (FBS)
Phosphate-buffered saline (PBS)
Trypsin-EDTA solution
Incubator set to 37°C with 5% CO₂
Day 1: Prepare Lentiviral Particles (if not purchasing pre-made)
- Transfection of HEK 293T Cells:
Seed HEK 293T cells in a 10 cm dish at approximately 70-80% confluency.
Prepare a transfection mix containing the lentiviral packaging plasmids, the lentiviral vector encoding hTERT, and a transfection reagent like Lipofectamine in serum-free DMEM.
Add the transfection mix dropwise to the HEK 293T cells and incubate for 6-8 hours.
After incubation, replace the medium with fresh, complete DMEM containing 10% FBS.
- Collect Lentiviral Supernatant:
After 48-72 hours, collect the lentiviral-containing supernatant from the transfected HEK 293T cells.
Filter the supernatant using a 0.45 µm syringe filter to remove cell debris.
Optional: Concentrate the lentivirus using ultracentrifugation or commercially available viral concentration kits, if desired.
Day 3: Transduce Primary Cells with hTERT Lentivirus
- Prepare Primary Cells:
Seed the primary cells (e.g., fibroblasts, epithelial cells) in a 6-well plate at 50-70% confluency in complete growth medium.
- Lentiviral Transduction:
Add 2-10 µg/mL of polybrene (a cationic polymer that enhances viral transduction) to the culture medium.
Add the filtered hTERT-containing lentiviral supernatant to the cells at an appropriate multiplicity of infection (MOI) based on the cell type.
Incubate the cells with the viral supernatant for 24 hours in a humidified incubator at 37°C and 5% CO₂.
- Medium Replacement:
After 24 hours, replace the viral supernatant with fresh, complete medium and continue incubation for an additional 48-72 hours.
Day 6: Selection of Transduced Cells (Optional)
- Antibiotic Selection:
If the lentiviral vector contains an antibiotic resistance marker (e.g., puromycin or neomycin), begin selecting the successfully transduced cells by adding the corresponding antibiotic to the growth medium.
Use puromycin at a concentration of 1-2 µg/mL for 3-5 days, depending on the sensitivity of your cell type.
Continue antibiotic selection until all non-transduced (uninfected) cells die, and only the antibiotic-resistant, hTERT-transduced cells survive.
- Expand Surviving Cells:
Once antibiotic selection is complete, allow the surviving hTERT-transduced cells to expand in fresh medium. Change the medium every 2-3 days until the cells reach 70-80% confluency.
Day 10+: Validation and Expansion
- Confirm hTERT Expression:
Validate the immortalization by confirming the expression of hTERT in the transduced cells using quantitative PCR (qPCR) or Western blotting.
Assess telomerase activity using the Telomerase Repeat Amplification Protocol (TRAP) assay, if desired.
- Test for Proliferation and Senescence:
Perform proliferation assays (e.g., cell counting or MTT assays) to confirm that the hTERT-immortalized cells have extended their proliferative capacity compared to control (non-immortalized) cells.
Stain the cells for β-galactosidase, a marker of senescence, to verify that the immortalized cells are not senescent.
- Expand and Freeze:
Once immortalization is confirmed, expand the hTERT-immortalized cells and cryopreserve them in freezing medium (e.g., 10% DMSO in FBS) for future experiments.
Notes and Considerations
Primary Cell Source: The success of immortalization depends on the source and type of primary cells. Some cells (e.g., fibroblasts) are easier to immortalize than others (e.g., neurons or myocytes).
Antibiotic Selection: If you use an antibiotic-resistant marker, optimize the concentration of the antibiotic for your cell type. Non-optimized concentrations can lead to either incomplete selection or cell death.
Verification: Always verify that the immortalized cells retain their original characteristics (e.g., morphology, differentiation potential) after immortalization, as some immortalization methods can alter cell behavior.
Alternative Immortalization Methods
If you are using SV40 Large T Antigen or HPV E6/E7, the procedure remains similar, but instead of introducing hTERT, you will use vectors that express SV40 large T antigen or HPV E6/E7 proteins. The following modifications apply:
Viral Vectors: Use plasmids or viral vectors carrying the SV40 large T antigen or HPV E6/E7 genes instead of hTERT.
Confirmation: After transduction, confirm immortalization by checking for the expression of SV40 T antigen or HPV proteins using PCR or Western blotting.
Final Thoughts
Immortalization allows for the generation of stable cell lines that can proliferate indefinitely, facilitating long-term studies in areas like cancer biology, drug screening, and tissue engineering. However, some immortalization methods can introduce genetic or functional changes, so validation and comparison with primary cells are important for accurate experimental outcomes.