AsianScientist (Aug. 31, 2018) – Consider this scenario: your current batch of cells just got contaminated by the new intern, and you’re about to revive the cell line by thawing out cryogenically frozen stock. The handwriting on the cryo-vial looks a little illegible and faded, but you think you can make out the word ‘HeLa’ (though your colleague thinks it looks like ‘HEK’).
The cells were frozen before you joined the lab as a PhD student, and the postdoc who performed the deed has since left for a presumably more lucrative career in a pharmaceutical company. How do you know if the cells you’re about to thaw are indeed what you think they are?
Bearing in mind that all your downstream experiments and data analyses will be based on the cell line in question, you’ll want to be very certain that you’re working with the right starting material. Otherwise, you could end up producing a paper that adds to the count of more than 32,000 research articles that involved cell lines of ambiguous origin and identity. So what checks can you carry out to authenticate the cells you work with?
Seek alibis and witnesses
The simplest way to find out if a cell line in your lab is correctly identified is to talk to colleagues who have worked with the cell line before and can vouch for its authenticity. Even then, don’t just take their word for it—ask to see some documentation; cell lines ordered from major cell banks such as ATCC typically come with a specification data sheet containing details on species origin, culture conditions, passage number and doubling time, among others.
Some of these data sheets even come with an image of the cells, providing you with an opportunity to at least verify cellular morphology. Having said that, while the spindle-shaped fibroblast may be easily distinguishable from the cobblestone-like keratinocyte, you’d need a highly trained eye to be able to tell apart a mouse fibroblast from a human one, or one human cancer cell line from another.
If you would like to hold yourself to a higher standard of verifying the cell lines used in your lab, then you’ll have to probe deeper.
It’s all in the genes
Just as forensics scientists make use of genetic material left at a crime scene to indict a perpetrator, some readily-available DNA tools in the lab can be used to authenticate cell lines.
Should you want to find out which species your cell line belongs to, and whether there has been inter-species contamination (e.g. the mixing of mouse and human cell lines), you could use species-specific primers and run a multiplexed polymerase chain reaction (PCR). After separating the PCR products using agarose gel electrophoresis, the fragment lengths will give an indication of which species the cell line belongs to. This is probably the least expensive and most convenient method for species identification.
Another more robust way to verify the species origin of a cell line is to perform DNA barcoding, which involves amplifying and sequencing a short fragment of a gene that encodes cytochrome c oxidase subunit 1 (CO1). You can then copy and paste the nucleotide sequence into this online database and—voila!—you’ll get a species ID.
Same same but different
However, neither PCR with species-specific primers nor CO1 DNA barcoding can help you distinguish HeLa cells from HEK cells, both of which are of human origin. This is where you’ll need a technique known as short tandem repeat (STR) profiling.
STRs, as their name implies, refer to short sequences of DNA that are successively repeated in specific regions of the genome. Because the number of repeats in each region differs between individuals, by analyzing 18 of such regions, a unique ‘DNA fingerprint’ can be produced for each person. Hypothetically, this means that STR profiling can pick out one person from a crowd of one million trillion people!
Because of its high discriminating power, STR profiling is the preferred protocol for cell line authentication, and several publicly–searchable databases have been established with reference STR profiles of known cell lines.
So there you have it—three approaches to find out exactly what kind of cells you’re working with. Addressing the problem of misidentified cultures is a bit of a hassle, but science will be better off for it.
This article is from a monthly column called Hacking a PhD. Click here to see the other articles in this series.
Copyright: Asian Scientist Magazine; Photo: Shutterstock.
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