Cell detachment with trypsin/EDTA: the biochemistry going on when you take cells off!

A lot of tissue culture (“TC”) work in the lab involves growing “adherent cells” - basically you grow cells stuck to dishes or plates. The cells need plenty of space (but not too much - they need some friends!) and plenty of media (food). Since the cells will keep replicating, making more copies of themselves, they’ll quickly run out of both (generally every few days depending on how dense you plate them originally). So, before they do (typically when they’re at ~80% confluency (covering 80% of the surface of the dish)) you detach them from the dish, dilute them in fresh media, and plate them in a new dish. We call this “passaging.” One of the ways we commonly detach cells is with a trypsin/EDTA solution.
blog version: bit.ly/trypsin_edta
Trypsin is a serine protease, a pretty generic protein chewer. MUCH more on trypsin and other proteases here: bit.ly/proteaseinhibitors; TH-cam: • Proteases and their in...
But the basic thing you need to know for now is just that it chews proteins. And you know what’s causing the cells to stick to the (specially-treated) surface of the dish and to one another? Proteins!!! So trypsin can sever these connections. But if you add too much trypsin and/or let it go for too long, trypsin can sever a lot more than just those… So you don’t want to over-trypsinize.
We therefore want to help make trypsin’s job easier, make those connections weaker to begin with. And this is where the EDTA comes in. EDTA (ethylene diamine tetra acetic acid) is a chelator, a compound that binds tightly to metal ions (charged particles), including calcium and magnesium. And why this matters is that some of the main adhesion-promoting proteins are calcium-dependent. It’s even in their name - CaDHerIns: Calcium Dependent adHerence proteIns. Binding to calcium causes these proteins to adopt a conformation (shape) that helps them bind to one another. Without that calcium, they don’t have the right shape for this. So EDTA helps destabilize the connections so trypsin can sneak in. Teamwork for the win!
You still need a way to stop the trypsin, though - and thankfully the media naturally has trypsin inhibitors! The media typically contains serum (often fetal bovine serum, FBS), the cell-less part of blood. Serum doesn’t have cells, but it does have a lot of other stuff - including a couple types of protease inhibitors that can incapacitate trypsin. The main ones at play (or I guess anti-play) are alpha-1-antitrypsin and alpha-2-macroglobulin). More on anti-trypsin in a minute cuz it’s pretty cool…
Since the media has serum, and serum has these inhibitors, you just have to add media and trypsin will cut it out (stop cutting your cells out!). Also, since the media has serum, and hence inhibitors, you need to make sure you thoroughly remove that media before you add the trypsin. And when you do that washing, you want to make sure you wash with a solution that doesn’t have calcium, but does have salts so you don’t send your cells into osmotic shock or anything (e.g. DPBS -calcium -magnesium).
Typically, the protocol is something like this:
- check cells under the microscope to see if they look like they need a passaging
- aspirate (suck off) old media
- wash with DPBS
- aspirate DPBS
- add enough trypsin/EDTA to coat surface (often a 0.05% solution)
- stick in incubator
- check in a couple min to see if detached (hold up to light and see if they’re still stuck or flowing nicely)
- check every 30s or so until they are
- as soon as they’re all detached, add media (about twice as much as you added trypsin/EDTA)
- pipet out the cell/trypsin/edta/media mix into a falcon tube & centrifuge (I typically do 1000g 5 min)
- aspirate trypsin/edta/media mix
- resuspend in smaller volume fresh media
- count cells
- calculate how much cells and how much media to plate in new vessels
- plate them
- stick them in the incubator
- check on them daily to make sure they’re happy
- when they start to get crowded, passage them again! (but not too many times - instead, when you get high up in passage numbers, thaw a new stock vial from an early passage). more on cryopreserving cell stocks: bit.ly/cryopreserving_cells TH-cam: • Cryopreserving cell st...  
finished in comments

[@thebumblingbiochemist]

now a fun note on anti-trypsin, which has a fun name - it’s a SERPIN. These are not snakes, they’re “serine scissor safety guards.” Their name comes from SErine PRotease INhibitors and they act as “fake substrates” that get stuck in the protease when it tries to cut them - it’s able to cut them but it can’t release them because, once cut, the serpin can (still bound to trypsin) shape-change, drag trypsin along with it, and move the trypsin’s active site out of whack in the process.
An example of a serpin you might have heard mentioned in commercials for emphysema treatments is alpha-1-antitrypsin - in the bloodstream, it protects tissues from the serine protease elastase, which a type of immune cells called neutrophils secrete into sites of inflammation to break down connective tissue so that blood cells have an easier time getting in to go to work repairing damage. alpha 1-antitrypsin acts as a sort of “moat” that limits elastase’s area of action so it doesn’t just go chewing up connective tissue throughout your body. But smoking can modify this serpin so that it fails to do its job adequately, so the moat is breached and elastase is able to chew up the lungs. Some people have a genetic alpha-1-antitrypsin deficiency, so they’re more at risk for emphysema even in they don’t smoke. alpha 1-antitrypsin is just one example of more than 30 serpins have been discovered in humans. A couple others are antithrombin, which keeps thrombin in check during clot formation and antiplasmin, which inhibits plasmin so that clots can be disassembled.
Here’s that cool page about cadherins: Cells Get Stick with Calcium, UIUC Theoretical and Computational Biophysics Group: www.ks.uiuc.edu/Research/cadherin/
Some helpful resources:
Subculturing Adherent Cells, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/subculturing-adherent-cells.html
Useful numbers for cell culture, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
Cell Dissociation Protocol using Trypsin, Millipore Sigma: www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-dissociation-with-trypsin
Evolution of cell culture surfaces, John A. Ryan, Ph.D., Corning Life Sciences: www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/evolution-of-cell
more on HEK293 cells: blog form: bit.ly/hekcells ; TH-cam: th-cam.com/video/tLtKMvZ2z5U/w-d-xo.html  
more about culture media: blog: bit.ly/cell_culture_media ; TH-cam: th-cam.com/video/8GKN60J9mC4/w-d-xo.html  
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@dennyhamrick2552]

Hey, I just wanted to say: I started my first tech job out of undergrad recently and your videos (and website content) have been unbelievably helpful, especially in understanding how each reagent in a process actually functions. Thanks so much for all the effort you put in; it is very appreciated

[@thebumblingbiochemist]

Soooooo happy to hear! Thank you so much. I really appreciate you saying that and wish you the best of luck in your new job!

[@alfredogonzalez8735]

When trypsinizing, once we add media and deactivate the trypsin, do we need to centrifuge and remove the media and suspend in fresh media? In my lab I've just been adding media, aspirating, and then adding half the volume to two different flasks that already have some fresh media. Just not sure if the centrifugation is necessary.

[@thebumblingbiochemist]

I always removed the trypsin-containing media so it didn't affect anything later on.

[@jennifer5nogueira]

Hi!! I'm a junior research in Brazil and I really love your work! One doubt: when culturing adherent cells, the tripsinization should only make them round and I finish the job by gently hitting the flask or let the tyrosine totally detach them from the bottom of the flask? I'm always afraid of letting them for a long period with trypsin, so I usually end up hitting the flask. Thanks!

[@thebumblingbiochemist]

I normally would do just like you