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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:
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.
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 |
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| Immortalized Cells |
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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 |
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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 |
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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.
| 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.
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.
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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!
