Molecular Minutes

Your Basic Guide to Cell Line Immortalization

Posted by Applied Biological Materials (abm) on February 12, 2020

how-to-make-immortalized-cell-lines-1

 

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 blog post we’ll go over:

  1. What are immortalized cells
  2. Strategies for generating immortalized cells
  3. An overview of the immortalization workflow
  4. Cell line quality control considerations

1. 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.

Figure 1: Immortalized Cells

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 blog post, 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

 

Have some immortalized cell lines but need new ones for your upcoming project?  Trade in your cell line with a new abm cell line through our free abmXchange program.


2. 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.
 
Figure 2: Normal vs. Immortalized Cells
 
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.
 

3. The immortalization workflow

Once you’ve decided what immortalization strategy is best for your project, there are two methods of performing the immortalization itself:

Method A: Plasmid transfection


One way to introduce the immortalizing agent (i.e. hTERT, SV40T antigen) into the cells is DNA transfection. DNA transfection methods include electroporation, lipofection, calcium phosphate, and more. abm carries many commonly used transfection reagents. Primary cells are less susceptible to DNA transfections and as such, many use the viral transduction method described below.

Method B: Viral transduction:


Primary cells are known to be difficult-to-transfection but receptive to recombinant viral transduction, especially adenoviral and lentiviral particles. The lentiviral transduction method, in particular, results in the stable integration of your gene of interest into the host genome for long term gene expression.

You can read extensively about the different viral transduction systems available on our comprehensive knowledge base.


Specific cell types require different reagents and methods. As mentioned earlier, many cell types require a combination of hTERT and viral oncogenes. For example, EBV is known for success in B or T lymphocytes, whereas Bmi-1 has shown success with nasopharyngeal epithelial cells, and HPV has been successful with keratinocytes. When in doubt, SV40 and hTERT are good options to start with. It is also advised to observe which pathway you are researching and choose the reagent that will not interfere with your current or future experiments. Always perform a literature review on similar cell lines to determine what will work best.


Here is a basic workflow to help you understand the key steps of a typical cell immortalization project:

 

Step 1: Seed the cells so that they are 50-60% confluent. Incubate overnight.

 

Step 2: Infect the cells with a variety of cell immortalization reagents (e.g. lentiviruses). Combinations can be tried if necessary.

 

Step 3: Remove virus from the cells and replace with fresh growth medium.

 

Step 4: Monitor the cell's growth rate and morphology. Grow infected cells in parallel with control cells to assess the proliferation rate.

 

Step 5: QC the candidates that show promise of immortalization. Keep cells in culture for up to 20 passages. Use qRT-PCR to test for transgene expression.

Figure 3: Basic immortalization workflow

Figure 3: Basic cell line immortalization workflow.

 


4. 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 blog post, 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 technical@abmgood.com or leave us a comment below! Good luck on your project!


References:


Ahmed N, Maines-Bandiera S, Quinn MA, Unger WG, Dedhar S, Auersperg N. Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium. Am J Physiol Cell Physiol. 2006 Jun;290(6): C1532-42. Epub 2006 Jan 4.


Ben-Porath I, Weinberg RA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol. 2005 May;37(5):961-76. Epub 2004 Dec 30.

Ben-Porath I, Weinberg RA. When cells get stressed: an integrative view of cellular senescence. J Clin Invest. 2004 Jan;113(1):8-13. Review.
Dimri G, Band H, Band V. Mammary epithelial cell transformation: insights from cell culture and mouse models. Breast Cancer Res. 2005;7(4):171-9. Epub 2005 Jun 3. Review.


Fridman AL, Tainsky MA. Critical pathways in cellular senescence and immortalization revealed by gene expression profiling. Oncogene. 2008 Aug 18.


Jha KK, Banga S, Palejwala V, Ozer HL. SV40-Mediated immortalization. Exp Cell Res. 1998 Nov 25;245(1):1-7. Review.


Kirchhoff C, Araki Y, Huhtaniemi I, Matusik RJ, Osterhoff C, Poutanen M, Samalecos A, Sipilä P, Suzuki K, Orgebin-Crist MC. Immortalization by large T-antigen of the adult epididymal duct epithelium. Mol Cell Endocrinol. 2004 Mar 15;216(1-2):83-94. Review.


Lundberg AS, Hahn WC, Gupta P, Weinberg RA. Genes involved in senescence and immortalization. Curr Opin Cell Biol. 2000 Dec;12(6):705-9. Review.


Matsumura T, Takesue M, Westerman KA, Okitsu T, Sakaguchi M, Fukazawa T, Totsugawa T, Noguchi H, Yamamoto S, Stolz DB, Tanaka N, Leboulch P, Kobayashi N. Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT. Transplantation. 2004 May 15;77(9):1357-65.


Rosalie Sears, Faison Nuckolls, Eric Haura, Yoichi Taya, Katsuyuki Tamai, and Joseph R. Nevins. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes & Dev. 2000 14: 2501-2514.


Stewart SA, Weinberg RA. Senescence: does it all happen at the ends? Oncogene. 2002 Jan 21;21(4):627-30. Review. Stewart SA, Weinberg RA. Telomeres: cancer to human aging. Annu Rev Cell Dev Biol. 2006;22:531-57. Review.


Thibodeaux CA, Liu X, Disbrow GL, Zhang Y, Rone JD, Haddad BR, Schlegel R. Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant. Breast Cancer Res Treat. 2008 Jul 20.


Yang G, Rosen DG, Colacino JA, Mercado-Uribe I, Liu J. Disruption of the retinoblastoma pathway by small interfering RNA and ectopic expression of the catalytic subunit of telomerase lead to immortalization of human ovarian surface epithelial cells. Oncogene. 2007 Mar 1;26(10):1492-8. Epub 2006 Sep 4.


Yang G, Rosen DG, Mercado-Uribe I, Colacino JA, Mills GB, Bast RC Jr, Zhou C, Liu J. Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells. Carcinogenesis. 2007 Jan;28(1):174- 82. Epub 2006 Jul 8.

 

Topics: Cell Culture, Cell line

Molecular Minutes

Educational resources for life scientists and interviews with scientists/science communicators in the field.

For more in-depth articles, check out our knowledge base, which covers topics such as CRISPR, Next Generation Sequencing, PCR, Cell Culture, and more.

Blog managed by Applied Biological Materials (abm). 

Subscribe to Email Updates

Recent Posts