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Your Basic Guide to Cell Line Immortalization

45 min Read
Summary Video

You’ve probably heard the saying, “all good things come to an end” - especially when it comes to precious primary cell cultures that reach senescence after a few passages. Thanks to the development of immortalized cell lines, however, this is no longer true! In this article we’ll go over:

  • What are immortalized cells
  • Strategies for generating immortalized cells
  • An overview of the immortalization workflow
  • Cell line quality control considerations
Telomeres and Cell Immortalization
What are immortalized cells?

You’re probably used to using cells that are taken directly from living tissue, called primary cells. The difficulty with primary cells is that their telomeres shorten after every cell division, causing the cells to enter senescence and stop dividing after only a few cell cycles. This means that if you are working on a long term project, you’ll frequently need to keep harvesting and re-establishing new batches of primary cells. In addition, every batch of cells is different due to different harvesting conditions, making reproducibility a headache!

Immortalized cells (also called continuous cells or cell lines) are primary cells whose telomeres and/or tumour suppressor genes have been altered. Tumour suppressor genes (e.g. p53 and Rb) are important for signalling the cell to stop dividing when the likelihood of DNA damage is higher (i.e. after multiple cell cycles, read more about the cell cycle on our knowledge base). In the case of immortalized cells, these genes have been knocked down or their function inhibited so that the cell is able to keep dividing indefinitely.

Telomeres and Cell Immortalization

Figure 1 – Telomeres are repetitive regions of DNA that form protective “caps” at the end of a chromosome, protecting the chromosome from deterioration. After each successive round through the cell cycle, these telomeres shorten, a process which eventually leads the cell into senescence where the cell stops dividing.

Ideal immortalized cells have genotypes and phenotypes similar to their parental tissues. Some labs use the same immortalized cells for decades, and consequently, their cell lines are well characterized and provide a consistent baseline for their long term projects. The oldest and most commonly used human cell line is the HeLa cell line, established from cervical cancer cells in the 1950s! But, as you’ll see in the quality control section of this article, it is important to check the cell line’s identity to ensure your cell lines are what you think they are.

  Advantages Disadvantages
Primary Cells
  • Similar chromosome number as parent tissue
  • Have specialized biochemical properties as parent tissue (growth factor and hormone secretion)
  • Best experimental models for in vivo situations
  • Finite lifespan/limited number of cell divisions
  • Considerable variation in population and between preparations
  • Difficult to maintain in culture (very “finicky”) – also more susceptible to contamination
  • Difficult to obtain (donor availability)
Immortalized Cells
  • Have a tendency to grow more quickly and can grow up to higher cell density compared to primary cells
  • Uniform cell type (mostly clonal)
  • Same donor source therefore less batch-to-batch variation
  • Most cellular characteristics are maintained from parent cell
  • Saves money and time for long term projects
  • Good for in vitro experiments
  • Cells may differentiate over time in culture
  • Cell behaviour in vitro may not represent in vivo situation
  • Potential alteration in phenotype or cell functions

Cell immortalization strategies

So, how do you engineer an immortalized cell line?

There are two major methods:

Method A: Telomerase Reverse Transcriptase protein (TERT) expression

The TERT protein is the catalytic subunit of the telomerase enzyme, and is normally inactive in most somatic cells. In this method, you can insert cDNA coding for the human telomerase reverse transcriptase (hTERT) protein into your primary cells of interest. When hTERT is exogenously expressed, the cell is able to maintain sufficient telomere length to avoid senescence. This is the most recently developed approach for cell immortalization.

Advantages Disadvantages
  • Works well for immortalization of cells that are most affected by telomere length (e.g. human cells)
  • Least likely to cause cancer-like phenotypes
  • Cell lines generated using this method have stable genotypes and retain critical phenotypic markers
  • May not obtain high success immortalization rate as compared to Method B for certain cell types
  • Overexpression of hTERT can even induce cell death in some cell types (e.g. epithelial cells)
  • This method, alone, may not be enough for successful immortalization

Method B: Viral oncogenes

Viral oncogenes such as the large T antigen from the SV40 virus or the E6/E7 oncogenes from HPV can achieve immortalization by suppressing tumor suppressor genes (e.g. p53 and Rb). This method takes effect quicker than Method A but may change some of the cells’ characteristics.

Advantages Disadvantages
  • Quickest route to immortalization
  • Useful for difficult-to-immortalize primary cells (e.g. epithelial cells)
  • May change the cell’s characteristics (e.g. loss of contact inhibition, genomic instability, disruptions of cell cycle checkpoints)
  • This method, alone, may not be enough for successful immortalization

In many cases, method A and B alone may not be enough for a successful immortalization. Recent studies have found that co-expressing hTERT with viral oncogenes have a higher success rate and result in more authentic and normal cell models with well-defined genetic backgrounds.

Typical lifespan of a normal cell

Figure 2 – Typical lifespan of a normal cell (green arrow). With every division, the telomeres shorten until it eventually reaches what is called senescence where the cell stops replicating and dies off. By over-expressing or inhibiting a particular gene (e.g. disrupting the p53 and RB pathway), we can reverse or inhibit telomere shortening to allow a cell to keep dividing without reaching senescence.

Reagent Mechanism Available formats at abm
SV40T Antigen Suppresion of p53 and Rb genes Lentivirus, Adenovirus, Retrovirus
p53 siRNA Knockdown of p53 tumor suppression gene siRNA Lentivirus
Rb siRNA Knockdown of Rb tumor suppression gene siRNA Lentivirus
Ras Suppression of Rb Lentivirus
C-myc T58A Suppression of p53 Lentivirus
Bmi1 Inhibition of p16/Rb pathway Lentivirus
CDK4 Suppression of p16/Rb pathway Lentivirus
HPV 16 E6/E7 Inhibition of p16/Rb pathway Lentivirus
EBV Viral oncogene -
hTERT Elongation of telomeres Lentivirus, Adenovirus, Retrovirus

Table 1: This table showcases common immortalization methods. Each mechanism involves disruption of key genes involved in the replication process shown in Figure 2. The rightmost column describes the formats available from abm’s collection of immortalization reagents. You can read more about the different viruses on our knowledge base.

An overview of the immortalization workflow
Basic cell line immortalization workflow

Figure 3 – Basic cell line immortalization workflow.

Cell line quality control considerations

Hurray! At this stage you have generated an immortalized cell line. But, before you start using your cell line in your projects, there are few important quality control checks you can do to ensure your cell line is in perfect condition.

A. Characterization of Your Cell Line

After every immortalization, you should always check your cells for the proper markers, as well as the cell’s functionality to make sure it represents the primary cells that you worked with. This step is especially important when you are studying cellular pathways because you would not want the immortalization step to have altered the specific pathway, function, or phenotype of the cells.

For example, if you plan to use your cells for a cytotoxicity assay, it is a good idea to perform a cytotoxicity assay with your immortalized cells alongside the primary cells to ensure your newly immortalized cells respond to stimuli in a manner comparable to their primary cell of origin.

B. Passaging and Transgene Expression Test

Passage your cells a minimum of 30 times to prove that the transgene has been stably expressed and to observe that their growth rate has improved and density capability has increased.

C. STR Profiling

As mentioned in the beginning of this article, it is of crucial importance that you verify the identity of your cell line as issues of cross-contamination or population mixing can occur. For example, according to a paper published in Science, the famous HeLa cells which are widely used in labs worldwide have been found to have contaminated many cell lines, including the Hep-2 and INT407 cell lines.

The gold standard for identifying cell lines is Short Tandem Repeat (STR) profiling. STR analysis is a method where short tandem DNA repeats at specific loci are compared to a standard reference profile. A cell line’s STR Profile can be used to confirm its identity and it is a good idea to do regular STR profile checks to ensure your cell line has not been contaminated over time.

In response to incidents of cell line mis-identification, funding agencies such as NIH now require cell lines used in grant proposals to have STR profiling analysis done, and many journals are beginning to encourage this crucial quality control check as well.

abm offers STR profiling Services that follow the International Cell Line Authentication Committee (ICLAC) standards.

D. Mycoplasma and Pathogen Detection

Finally, it is always a good idea to test for microbial contaminants such as fungus, bacteria and mycoplasma.

Mycoplasma, in particular, is one of the most common contaminants in cell culture laboratories. It is often difficult to detect Mycoplasma through the typical visual inspection under the microscope. However, this bacteria can affect the cell’s proliferation, change its gene expression profile, and other effects that can skew your experimental results. There are many kits available, such as abm’s PCR Mycoplasma Detection and Elimination kits, that can easily detect and remove over 50 types of Mycoplasma from your cells.

We hope this article has helped give you the confidence you needed to start on your first cell immortalization project! If the immortalization process sounds like too much of a hassle, don’t worry - there are many cell line repositories available, including abm’s collection of 700+ immortalized cell lines and unique cell lines for drug discovery, available worldwide. You can view our catalog or request a free physical copy here. Don’t see a particular cell line that you are looking for? Feel free to inquire with us as we are constantly adding new cell lines to our repertoire.

Have questions or need help with your project? Contact our Technical Support team at [email protected]. Good luck on your project!

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