I like to know whether it is possible just to wash out trypsin with PBS, centrifuge followed by resuspending the pellet with serum free medium for cells to be cultured with serum free medium?
Hi, it’s necessary to use FBS neutralization trypsin when you want to Passaging Cells. Because the trypsin will make the cells detach and have a viability hurt to them when there is residual trypsin, that FBS will neutralization this reaction. If the cells need to culture with serum-free medium, you need to resuspend the cells in serum-free medium.
A buffer containing calcium and magnesium ions will also slow down trypsin activity. FBS is however the more effective way to inhibit trypsin activity. If you are incubating in serum-free medium after trypsin treatment, you can inhibit with FBS containing medium and follow your first centrifugation step with an additional wash step using serum-free medium.
If the goal is to avoid contaminating the serum-free medium with serum proteins, I wonder whether the trypsin can instead be inactivated by addition of a trypsin inhibitor, such as PMSF or BPTI.
Hi Adam, I cannot find supplier for PMSF and BPTI. Normally people use soyabean trypsin inhibitor but it is expensive. How about your two products? Are they inexpensive? Hope the two will not do permanent inhibition because I still need to use trypsin to detached cells from flasks and they inhibit trypsin.
Phenylmethylsulfonyl fluoride (PMSF) is an inexpensive inhibitor of serine proteases. The question is, how toxic is it to the cells?
Here is a page from Sigma-Aldrich on trypsin inhibitor proteins and their uses in cell culture.
http://www.sigmaaldrich.com/technical-documents/articles/biology/trypsin-inhibitors.html
Dear Jamail, our purpose is to deactivate the trypsin after the cells detachment, so that it wont hinder cells attachment later on in our successive passage. Trypsin is an enzyme (serine protease) so how come any buffered saline of neutral pH has any effect on an enzyme? rather it will simply dilute somewhat your trypsin or slows it down may be, but the catalytic activity of trypsin will still be there no matter how much PBS washing you will use. It wont be 100 % free from trypsin. So you definitely need proper trypsin inhibition.
I think it is highly possible of just washing with PBS. If we are worry about residual amt of trypsin, just wash twice before culturing them with serum free medium. Since washing twice, there will not be enough amt of trypsin left over and even if left minimum amt, it will be further diluted with serum free medium and they are no harm at all. I have done and successful. No issue.
Why don't you just use something like this?
https://www.promocell.com/product/trypsin-neutralizing-solution/
If you do the resuspension/centrifugation/aspiration twice, this should generally work. After just one iteration, there is still some inhibition of growth after plating in a serum-free media.
Mingchuan Yu, good question, and thank you for giving an update on what worked for you.
Michael Koksharov, thank you for sharing this invaluable experience as well.
BUT alternatively and in support of what Adam B Shapiro said, if you are avoiding the contamination of your SFM with serum proteins, you can dissociate your cells with TrypLE™ reagents.
TrypLE™ is a recombinant enzyme that does not require neutralization, inactivation, or washing off. According to their product descriptions, dilution alone (with serum-containing or serum-free media) inactivates the TrypLE™ enzyme, with no need for trypsin inhibitors.
I have not used this personally, but their testimonials look good and are reasonably priced. You can check out the links below if you want to try them out.
https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html
https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express/tryple-testimonials.html#:~:text=TrypLE%20is%20easy%20to%20use,and%20use%20compared%20to%20Trypsin.%22
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