Cell Immortalization: How to Immortalize Cells

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! Still have questions or want a specific video topic covered? Leave them in the comments below! Please like and subscribe if you found this video helpful

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