Wash cells or not after trypsinization? - (May/03/2005 )
I believe trypsinization is done to disrupt the cell matrix and make them float. As all the cells would be now floating in the trypsin media, i feel theres no need to wash, coz only 1% cells would be left in the flask adhering.
The purpose of washing is not to wash cells off the surface but to get rid of trypsin.
i was trying to separate out the cells after trypsinisation. But unfortunately evenafter 2000 rpm 10 mnts.. cells were still in the suspensiion (clump).
could anyone help me to solve this issue. ( I used 10 ml of trypsine/versine for 75 cm2 flask) soln and incuated for 10 mnts
Hi, I have found that to keep my cells from clumping after trypsinization I first resuspend the cells in the plate in the small volume of trypsin solution by pipetting up and down with 1 ml tip until I cannot see any more cell clumps, then adding the cells directly to a few mls of MEDIA in a 15 ml tube, and they stay non-clumped --> then I centrifuge to wash. If I add them to PBS or another solution while they are in trypsin they tend to clump up.
Also it may be helpfull to reduce the time and rpm of centrifugation to avoid clumping: 1000 rpm for 5 minutes may be sufficient to pellet the cells, and make then easier to resuspend. Also, once they are pelleted, it is helpful to resuspend in no more than 1 ml media by pipetting up and down, and NOT by vortexing. It is harder to resuspend cells by vortexing, or if the volume you are trying to resuspend in is too large. Once they are resuspended in a small volume, then add the rest of the media necessary for replating.
Also about trypsinizing in general: I work with IMCD3, a very tough cell line that adheres strongly. My boss has instructed me to leave the cells in PBS for a total of 45 minutes, replacing the PBS 3 times (every 15 minutes). Simply leaving the cells in PBS for awhile does loosen them. I typically only do 2 x 15 minutes in PBS depending on how much time I want to take, and you will see that after even just 15 minutes some cells are starting to round up, and after 30 minutes some cells may be loose (more or less depending on your cell line I guess). Then, I add about 1 ml trypsin (0.25%) diluted with 1-2 mls of PBS (so that the solution can cover the area of the cells more evenly and to conserve trypsin), then put in the incubator for no more than 5 minutes. Sometimes after 30 minutes in PBS the cells are already quite loose and simply pipetting them and rinsing the plate with the trypsin solution will get them off, no incubation necessary. Pre-loosening with PBS also conserves trypsin as you need less to get the cells off.
Since my boss has instructed me to wash off the trypsin that is what I do, adding the cells resuspended in trypsin to some media, spin for 2k rpm 5 minutes, then resuspend in 1 ml media. Pipetting the cells to resuspend is also harsh on them, so if you have a delicate cell line this may not be advised, or try to pipet more slowly.
Trypsin is a protease, and cleaves any proteins at lysine and arginine residues: while it is used in this case to release the cells from their protein attachments to the plate, it also acts on any extracellular proteins, so it may be toxic to some cells because it cleaves signaling or other external proteins the cells need to survive, and so may kill them if they are left in it too long.
FBS has protease inhibitors that inactivate the trypsin, which is why adding the cells in trypsin to media containing FBS is enough to inactivate the trypsin and they can be directly replated without washing in most cases (which is what I used to do without problem).
However if your cell line is a primary one (recently out of a living organism) or otherwise delicate, it would be best to minimize their exposure to trypsin and to wash them after trypsinization (in case not all trypsin is deactivated by the FBS in the media); and maybe also if your testing involves analysis of external proteins (like Flow Cytometry) washing is probably a good idea.
I use Primary human foreskin cell line and I never washed the cells after trypsin treatment. Usually after adding trypsin, I spread the trypsin on the monolayer by rocking the flask gently with hand and then remove the trypsin I added. I then incubate for couple of minutes and when the cell detach I add serum containing medium and proceed for subculturing. (for some cell line removing the trypsin does not work esp. the rat kidney cell lines).
TripLE Express is a GREAT alternative to Trypsin!!! I do not wash, thus minimizing centrifugation harm; and TripLE Express is MUCH LESS HARSH on cells than Trypsin. For me it seems to be working just as well at detaching cells. I've also noticed that I have less clumping of cells.
I add TNS (Trypsin Neutralizing Slotion) after detaching the cells with Trypsin.
I never give a wash step after trypsinization. Just add medium (with serum) double the amount of trypsin, serum instantly neutralizes the tryspin. and then I take 100-500ul for subculture depending on the use. I use 1-2ml for 100mm dish. works fine...
In the past, I neutralized the trypsin with bovine serum, and the cells were very happy. Now, though, our lab switched to a serum free medium to prevent differentiation of stem cells, so they need to be washed (since there is no serum in this medium to neutralize the trypsin). They are not nearly as happy anymore.
Are the Caco-2 cells include at this? they have a strong tendency to form clumps...
SafariMelissa is the only one in this entire thread who has mentioned ALTERNATIVES to TRYPSIN. If you want single cell suspensions for whatever reasons then use trypsin. Trypsin is the tried and tested enzyme to use. However on a scale of 1-10 it is a 10, in relation to what damage it can potentially do to your cells. It strips surface receptors and can be internalised causing damage to intracellular proteins.
Other enzymes which are less harsh can be used as an alternative to Trypsin, causing less damage to the cells. In general you would use these on primary's as most cell lines are fairly resistant to trypsin. They are all inactivated in the same way as trypsin, using FCS/FBS. These include :-
Collagenase's : Types I, II, III and IV.
If you are having trouble with your cells and you think it is the trpysinisation which is causing the problem, try an alternative.