Worried your cells might die before the end of your project?
Immortalized cells have an infinite lifespan and are the perfect solution
for complex experiments or long-term projects.
In this video we'll discuss what immortalized cells are
the basics of the immortalization process
and cell line quality control considerations
Scientists often take cells directly from living tissue called primary cells for in-vitro research
However, in some cases, the telomeres of primary cells
shorten after every cell division eventually causing the cells to stop dividing
In other cases, tumor suppressor genes involved in cell cycle signaling
such as p53 and RB, prevents cell proliferation when DNA is damaged
When performing research, this can be a problem since your cells could reach
senescence before the end of your experiment
Ideally, you want your cells to proliferate indefinitely and to avoid batch to batch variation
such as differing phenotypes and genotypes
Well, this is exactly where immortalized cells can help!
So how do we create them?
In short, immortalized cells are primary cells
whose telomeres and/or tumor suppressor genes have been altered
When targeting the telomeres, insert cDNA into your primary cells coding for the hTERT protein
which induces telomerase activity and regenerates the telomeres
thus preventing replicative senescence
This method is simple and causes little change in the cell's characteristics
but could have low immortalization success rate
When targeting the tumor suppressor genes, inactivate them by inserting
viral oncogenes, therefore allowing the cell cycle to continue
This method takes effect quicker
but in some cases may change the cell's characteristics
Now, there are two methods of performing the immortalization itself
The preferred method is through lentiviral transduction as this will confidently
integrate the DNA into the host cell and stably express the gene of interest
The other method is through a plasmid transfection, which although it has a
lower success rate, may work for cells that are resistant to lentiviral transduction
So which reagent and method should you use?
Well, this depends on your specific cell type
Take primary human cells for example
inserting the TERT gene is usually a good reagent to use
since the hTERT protein naturally occurs in human cells
However, over expression of hTERT in some cell types
especially in epithelial cells, fails to induce cell immortalization and may induce cell death
Furthermore hTERT alone does not always suffice
many cell types require a combination of both methods
For example, recent studies have found that
co-expression of the hTERT catalytic subunit with either the p53 or RB siRNA
can immortalize human primary ovarian epithelial cells
To find out what will work best for your project, perform a literature review
to know how similar cell lines have been previously immortalized
Before performing your immortalization
make sure your primary cells are contamination-free
Standard tests consist of mycoplasma and pathogen detection
abm offers the necessary products and services
Once you confirm your cells are contamination-free, insert your immortalization reagent
through a lentiviral transduction or a plasmid transection
Get in touch with us if you need help with any step of the process or
if you would like our team of experts to perform the immortalization for you
while you focus on different aspects of your research
Finally, to verify your cells were immortalized
passage them alongside your primary cells
and observe their continuous proliferation over time
abm considers cell lines that last 20 or more passages as immortal
If applicable, you can also test for GFP fluorescence or antibiotic resistance
Always check the cell morphology microscopically
to ensure your cells look healthy and as expected
Lastly, perform an STR profile to make sure there was neither cell contamination
nor population mixing
This will also check your cell lines ID by
verifying the DNA short tandem repeats to a standard
This check is becoming an increasingly common requirement for publishing your research and journals
ensuring your results are accurate and reproducible
At abm, we offer all the necessary quality control services and products
we also have the world's largest collection of immortalized cells as well as custom cell line services
Check out our 'abm exchange' program to find out how to get a cell line or
other lab materials for free!
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