I am growing hCMEC/d3 cells in Lonza EGM media with Bulletkit supplements. The passage protocol I use calls for just directly adding the resuspended cells, trypsin (.25% in EDTA) and all, to the new media. Recently, I did a little experiment and have been centrifuging the cells down after trypsinizing, and then resuspending, in order to remove the trypsin, and I was surprised to see significantly accelerated growth. Has anyone else experienced this phenomenon?
That is an interesting observation. The serum that you add to the growth medium contains a trypsin inhibitor, so usually, it is assumed that all the trypsin is inactivated after resuspension in fresh growth medium.
Hello Ari,
I don't use the cells you mentionned but I have experienced with several mouse and human cell lines or primary neural stem cells.
First it depends if you are using serum or not in your medium. If you use serum, it will counterpart trypsin activity and just a 5 to 10 fold dilution could be enough to abolish the effect of trypsin and do not interfere with growth rate.
If you are working with serum-free medium the dilution to switch off the effect of trypsin will not be compatible with a proper concentration for your cells to re-growth.
Personnaly I always spin my cells to avoid any effect of trypsin by replacing with fresh medium. If I could not spin them I dilute and I use .05% trypsin in EDTA or ice cold EDTA in Ca free PBS to pass my cells.
I am not surprise at all that maintaining tryspin in your media could perturb growth rate.
Thomas
It depends on cell lines , mouse cells and few human cell lines I worked with were ok with trypsin left with 10-20 fold dilution. However, I realized over the time the cells needed longer incubation with trypsin to get de-attached, may be that was because of small amount of trypsin left in flasks which may have led the cells to adapt accordingly. So I would recommend same as Thomas, to be on safe side, spin the cells for their good.
Usually upon addition of serum, the trypsin is inactivated. But i don't know if it can have an accumulative effect on the growth pattern of your cells. I personally always spin down my cells (100 xg, 5 mins) after trypsinization to remove the supernatant.
If growth slows down, you can always add extra serum to the media
When I was used trypsin or accutase to detach my cells, I every time centrifuge pellet and add fresh medium to the cells.
In a situation when I use 0,5 mM EDTA I don't centrifuge cells during the passage.
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