What the HEK are HEK cells? They’re an “immortalized human cell line” that allows scientists to carry out fundamental research (drug testing, protein production, etc.) in human cells grown in dishes or flasks. If you take most cells and try to get them to grow in a dish, they won’t go for it – or maybe these “primary tissue cells” will divide once or twice if you feed them, but then quit unless you coax them with tons of growth factors, etc. But there are also some cell lines that are “immortal” – naturally or artificially – meaning they’ll keep growing and replicating (copying their DNA) and dividing, making more and more cells as long as you take care of them*. And HEK293 is one of the main cell lines along these lines (no pun intended).
note: Another common immortalized cell line is HeLa cells, which have an ethically-fraught path, being taken from a Black woman named Henrietta Lacks during a biopsy and used without her or her family’s consent – more on them here: http://bit.ly/henriettalacksstory
Speaking of ethically-fraught, let me just first address the elephant in the room. “HEK” stands for Human Embryonic Kidney cells and they originated from kidney tissue of a naturally-miscarried or aborted embryo in 1973 (the exact source is unclear).
The original HEK cells were isolated by a Dutch scientist named Alex Van der Eb, and then they were further adapted. HEK293 cells have a lot of mutations, which make them so good at growing. But they didn’t start with all of them. Instead, to get them to grow a bunch from the start, they were transformed with sheared DNA from Adenovirus 5 (Ad5). Well, that’s not *why* that transformation was done – instead, the scientists were trying to determine how some viruses could cause cancer – which is uncontrolled cell growth, hence the connection. https://www.liebertpub.com/doi/10.1089/hum.2020.29116.oxg But what is that “transformation” anyway?
Basically, a postdoc (recently-PhD-ed scientist) named Frank Graham got the cells to take in bits of Ad5 DNA. And some of those bits of DNA got integrated into the cellular genome (basically the viral DNA hopped into the human DNA). A particularly helpful hop for scientists was that, in his 293rd experiment, a ~4kbp (4,000 DNA base pairs long) stretch of Ad5 inserted into chromosome 19. And this stretch contains viral proteins called E1A & E1B, which interfere with cellular growth control mechanisms. Cells normally keep tight control over when they divide, and they undergo a programmed cell death called apoptosis if things get out of control. But the E1 proteins prevent this growth & quality-control system from doing its job, allowing the cells to keep growing. More here: https://www.nature.com/articles/ncomms5767
This allows them to grow “indefinitely”*
*Because they keep making more copies of themselves, you need to keep giving them more room to grow, in addition to more food. Therefore, every couple of days you “split” (aka “passage”) the cells – basically swap out the media & take some and put them in a new dish/flask. These cells typically have a bunch of mutations already and, each time they replicate, there’s the potential for more to arise. Therefore, to avoid mutation accumulation, you typically don’t want to passage them too many times. Instead, you can freeze “stock” tubes of an early passage and then thaw them out and “start anew” passage-number-wise once you’ve reached too high of passage numbers.
HEK293 are traditionally grown as “adherent cells” meaning you grow them stuck to a plate or a dish or the base of a flat flask. To change their food you can suck up (aspirate) the old media with a pipette attached to a vacuum line, and add new media. To split them, you add trypsin, a protease (protein cutter) which will help break up the cell-cell and cell-plastic sticking, then collect the cells, count them and then calculate how many to “seed” on a new plate/dish/flask, allowing them plenty of room to grow.
HEK293 cells can also grow as suspension cells (similar to how you can grow flasks of bacterial cell culture). Since they’re floating in their media, if you want to replace it, you first have to spin down the cell/media mixture in a centrifuge – since the cells are heavy, they’ll “pellet out” and you can then remove the old media, count the cells, add new media, etc. In the “lazy version”. you can also just count the cells in the old media, then dilute them in a bunch of new media to the desired concentration.
This suspension growth route, combined with HEK293’s transfectability has made these cells one of the workhorses of mammalian recombinant protein expression – basically scientists who want to study a protein can stick the genetic recipe for making that protein into these cells and get the cells to make it for them. It’s more complicated (and way more expensive) than expressing proteins in bacteria (or even insect cells), but it will give you the most naturally “human-like” proteins, in terms of proper post-translational modifications.
Basically, thanks to the universal nature of the genetic code bacteria and insect cells might be able to translate a human protein (piece together all the amino acids in the right order), but those proteins might not fold properly without the help of human-specific chaperone proteins to help with the folding. Even if they do fold, those proteins might not get the correct phosphorylations, glycosylations, etc. All these post-translational modifications, which can be crucial to a proteins’ functioning, get added by enzymes that the bacteria don’t have and that insect cells have different versions of. So, insect cells are generally better than bacterial cells for making human proteins, but if you want really authentic human-like, you have to turn to a mammalian cell line – and the two that are commonly turned to are HEK293 and CHO (Chinese Hamster Ovary).
There are a few common cell lines derived from the original HEK-293 cells:
- especially good for transfection (sticking genetic instructions inside them):
- HEK-293T
- these are great for transfection, but that’s not what the T stands for – instead, the T is because they express a temperature-sensitive version of the T antigen from the virus SV40. This “T antigen” is what normally allows the virus to replicate, so if you stick in a plasmid containing an SV40 origin of replication (ori), that plasmid will get replicated and thus passed on when the cell divides
- it also allows them to be used to produce virus for gene therapy, as well as viruses for transducing other cells (using a virus to inject genetic information into other cells). Basically, you can take a modified virus that lacks the T antigen and therefore can only replicate in these cells. And you grow it up in these cells. And then you get it to infect other cells. It can infect those cells, but can’t replicate in them, so can’t “spread” or be maintained, but can get those cells to express a protein of interest
- these are great for transfection, but that’s not what the T stands for – instead, the T is because they express a temperature-sensitive version of the T antigen from the virus SV40. This “T antigen” is what normally allows the virus to replicate, so if you stick in a plasmid containing an SV40 origin of replication (ori), that plasmid will get replicated and thus passed on when the cell divides
- T-REx cells
- have a tetracycline repressor gene, which allows you to control the expression of genes under a Tet promoter. When the cells are grown in the absence of the antibiotic tetracycline (Tet), that protein won’t get made. But when you add Tet, you will “induce expression” of the gene and the protein will get. This is good for controlling the expression of proteins that are toxic to the cell, and/or investigating the function of a protein at particular developmental timepoints
- HEK-293T
- HEK-293S – adapted for suspension growth (more on this below, but basically they’re able to grow suspended in liquid rather than stuck to plastic)
- 293SG
- good for generating homogeneously N-glycosylated proteins (proteins where all the sugar chains (glycosylations) that are added are the same from protein to protein – especially helpful for structural biology investigations of membrane & secreted proteins where heterogenous glycosylations can make it hard to see what’s going on)
- this cell line comes from random mutagenesis of the 293S line with a mutagen called ethylmethanesulfonate (EMS) – a mutation in the MGAG1 gene (an N-acetylglucosaminyltransferase) leads them to primarily produce Man5GlcNAc2 N-glycan modifications
- there’s also a version which expresses a stable tetR repressor, allowing it to be used for Ted-inducible protein expression
- especially good for recombinant protein expression:
- FreeStyle 293
- Expi293
- more about these here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression.html
Some technical notes about their growth & working with them:
- because they contain the adenoviral DNA, you have to take special safety precautions when working with them – “BSL2” (biosafety level 2) protocols
- and you also have to take precautions to avoid infecting them! (you don’t want the culture to become contaminated so you have to maintain sterility in your hood-work and potentially add antibiotics)
- the cells are “hypotriploid” – basically they have ~3 copies of each chromosome, with ~64 chromosomes per cell (once you mess with the cells’ quality control systems, chromosome numbers can vary greatly!) https://www.atcc.org/products/crl-1573
- you typically grow them at 37°C in 5% CO₂
- they double ~every 34-36h https://www.synthego.com/hek293
- for splitting, this Synthego site recommends “Splitting HEK293 cells should always occur during the log (growth) phase, at around 80-90% confluency or between 6 x 104 and 7 x 104 cells/cm2. Detach adherent cultures using Trypsin or TrypLE, aspirate by pipetting pre-warmed growth media gently over the back wall of the flask, then seed at 2 x 104 to 4 x 104 cells/cm2.” https://www.synthego.com/hek293
helpful sources:
Lin, YC., Boone, M., Meuris, L. et al. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun 5, 4767 (2014). https://doi.org/10.1038/ncomms5767
Yuan J, Xu W. W, Jiang S, Yu H, Poon H. F. The Scattered Twelve Tribes of HEK293. Biomed Pharmacol J 2018;11(2). Available from: http://biomedpharmajournal.org/?p=20696
Live and Let Live: The Remarkable Story of HEK293 Cells.Human Gene Therapy.Apr 2020.485-487.http://doi.org/10.1089/hum.2020.29116.oxg
ATCC entry on 293[HEK-293] (CRL-1573): https://www.atcc.org/products/crl-1573
ThermoFisher “Mammalian Transient Protein Expression”: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression.html
GenoFab, HEK293: An essential human cell line with a unexpected origin https://blog.genofab.com/hek293-cell-line
Synthego, HEK293 Cells: Background, Applications, Protocols, and More: A Guide to One of the Most Commonly Used Cell Lines https://www.synthego.com/hek293
more about various forms of recombinant protein expression: http://bit.ly/proteinpurificationtech
more about viral vectors: https://bit.ly/adenoviralvectors
