Immortalized Cells: Break free from the "lifespan limitations " of traditional cells and unlock endless possibilities for scientific research.

In in vitro cell experiments, a stable and reliable supply of cell resources is essential for various types of research. However, conventional cells tend to age over time during subculturing and eventually lose their ability to divide. This phenomenon is known as the "Hayflick limit" (under normal in vitro conditions, cells typically divide only 50-60 times). The advent of immortalized cell technology has introduced cells that can be subcultured indefinitely while maintaining stable performance, making them a valuable resource for scientific research. In this edition of the Cell Culture Academy, we will guide you through the fundamental principles and processes involved in creating immortalized cells and explore new frontiers in cell research together.

Ⅰ. Cellular Immortalization
Cellular immortalization refers to the process by which cells acquire the ability to proliferate indefinitely during in vitro culture by bypassing the Hayflick limit, either through spontaneous mutations or external interventions. These cells often retain key characteristics of the original primary cells and, under normal conditions, do not spontaneously become cancerous without additional carcinogenic mutations. However, genetic changes may still occur during prolonged culture, so it is important to continuously monitor their genetic stability.

Principle

Cellular immortalization primarily involves introducing exogenous genes into target cells to bypass the natural aging process and enable them to proliferate indefinitely. Two key genes commonly used for this purpose are the SV40 large T antigen (TAg) and human telomerase reverse transcriptase (hTERT).

1. SV40 Large T Antigen (TAg)
It is believed that the SV40 large T antigen binds to the tumor-suppressor protein p53 and its related proteins to form a complex, which inactivates the p53 protein. This inactivation disrupts the cell-cycle regulatory mechanisms and prevents the p53 tumor-suppressor signals from functioning. As a result, cells avoid entering the aging or apoptosis pathways, allowing them to proliferate continuously and achieve immortalization.

2. Human Telomerase Reverse Transcriptase (hTERT)
Telomeres play a crucial role in maintaining chromosomal stability. They shorten with each cell division, and when they reach a critical length, cells initiate the aging process. hTERT is the core catalytic subunit of telomerase, which uses telomeric RNA as a template to extend the telomeres and preserve chromosomal integrity. In normal cells, hTERT activity is tightly regulated by epigenetic and transcriptional mechanisms, preventing it from maintaining telomere length. Therefore, activating hTERT is a key strategy for achieving cellular immortalization.

Technical Implementation Path: How to Deliver "Special Instructions" into Cells?
The most common method for achieving cellular immortalization involves stably integrating the gene sequences for TAg or hTERT into the target cell genome using lentiviral or other retroviral vectors, enabling continuous expression. This approach effectively bypasses the cell aging mechanism, granting cells the ability to proliferate indefinitely and significantly extending their lifespan in vitro.

We’ve discussed the advantages and limitations of the lentiviral vector system in our article "Mastering Fluorochrome-labeled Cells: From Principles to Engineering." Interested readers can refer to it for more detailed information.

Application Areas

Thanks to their ability to proliferate continuously and maintain relatively stable biological characteristics, immortalized cells are widely used in fields such as basic research, biomedical development, and cell engineering. Common applications are summarized in Table 1.

Table 1: Applications of Immortalized Cells

Ⅱ. Simple Process for Constructing Immortalized Cells

1. Cell Line Selection and Status Confirmation

Lentiviral Packaging Cells: Choose HEK-293T cells for lentiviral production. These cells should be in the logarithmic growth phase and in good condition.

Target Cells: Select the target cells, ensuring they are also in the logarithmic growth phase and healthy. Refer to the construction methods for THLE-2 and SV-HUC-1 cell lines.

2. Lentiviral Vector Preparation

Co-transfect HEK-293T cells with plasmids containing TAg or hTERT, along with puromycin resistance markers, packaging plasmids, and envelope plasmids to generate recombinant lentiviral particles carrying the target genes.

3. Lentiviral Infection

MOI Optimization: Perform preliminary experiments to determine the optimal multiplicity of infection (MOI) that balances high infection efficiency with minimal toxicity.

Cell Culture: Harvest the target cells, create a cell suspension, and count the cells. Dilute the suspension to 1×105 cells/mL. Inoculate 500 μL into a 24-well plate and culture overnight at 37℃ with 5% CO.

Infection Procedure: Thaw the lentivirus on ice. Replace the original medium with fresh medium, then add the lentivirus at the optimal MOI. Add polybrene (final concentration: 8 μg/mL) to enhance infection efficiency. Incubate at 37℃ with 5% CO2 for 48 h.

4. Resistance Screening

Antibiotic Sensitivity Testing: Conduct a preliminary experiment to determine the minimum lethal concentration of puromycin. This concentration will be used for the subsequent screening.

Screening Process: After 48 h of infection, replace the medium with fresh complete medium containing puromycin and continue culturing. Refresh the puromycin-containing medium every 2-3 days, continuing until stable, resistant cells emerge.

5. Cell Identification

Gene Expression Verification: Use qPCR to confirm the expression of genes related to immortalization.
Cell Function Assessment: Assess the cell senescence status using β-galactosidase staining; continuously passage the cells and monitor their ability to surpass the Hayflick limit.
Phenotypic Stability Verification: Use immunofluorescence to detect the expression of specific marker proteins and evaluate the stability and purity of the immortalized cells.

Ⅲ. Precautions
1. During the virus packaging process, the quality and purity of nucleic acids play a crucial role in transfection efficiency. It’s recommended to maintain the plasmid concentration between 400 and 1,000 ng/μL, with a purity (A260/280 ratio) between 1.8 and 2.0.

2. To avoid significant reductions in virus titer, minimize freeze-thaw cycles when handling the virus. It’s best to aliquot the virus based on the amount needed for each use. Additionally, if the virus has been stored for more than six months, remeasure its titer before use to ensure optimal infection efficiency.

3. For cells that are difficult to infect, such as dendritic cells (DCs), consider performing multiple infections. For example, after the first infection (24 h), replace the medium with fresh virus and perform a second infection. This approach can significantly improve infection efficiency.

4. Immortalized cells can exhibit significant differences compared to primary cells in terms of proliferation, cell cycle regulation, telomerase activity, genetic stability, phenotype, and function. These changes provide unique advantages for experiments (such as indefinite proliferation and high stability), but may also limit their applicability in some studies (such as simulating physiological conditions). Therefore, when choosing between immortalized and primary cell models, carefully evaluate the suitability based on the specific experimental goals.

5. In theory, immortalized cells can be passaged indefinitely since they’ve overcome the Hayflick limit, with telomerase activity maintaining telomere length and preventing replicative senescence. However, over long-term passaging, genetic instability may lead to the accumulation of mutations, which could affect their phenotype and function. Furthermore, factors such as the composition of the culture medium, serum quality, and passaging methods can influence the cell state. It’s also important to note that different cell lines have varying levels of stability. Some immortalized cells may experience phenotypic changes or a decline in characteristics over long-term culture.