How to detach adherent cells when trypsin is ineffective?
May 18, 2009 12:15 PM   Subscribe

How would you detach primary adherent tissue cells from tissue culture polystyrene if trypsin/EDTA is ineffective?

I'm culturing primary adult human stem cells on tissue culture polystyrene. Some of my flasks have contained cells for 16 days, and 0.05% trypsin / 0.53 mM EDTA is now ineffective at detaching them.

(The cells are at passage 10, on their way to or at senescence, are now very slow-growing, and have not yet become confluent. Previous passages became confluent every eight days with a 1:4 split, and showed no problems with trypsinization.)

I'd like to run a final experiment on this line; any suggestions on detaching the cells? I have trypsin, EDTA, versene, and accutase here, but only two flasks left, so I hope to get recommendations on the best approach rather than just trying something and hoping for the best.
posted by Mapes to Science & Nature (8 answers total) 2 users marked this as a favorite
I've found that if T/E doesn't work (especially when pre-warmed to 37C), then accutase and versene and EDTA alone won't work. You can gently scrape the cells, or try to trypsinize @ 37C for 5-10 minutes and then mechanically dissociate (shaking or banging T-flask with heel of hand).
posted by scblackman at 12:19 PM on May 18, 2009

Best answer: I would try a higher concentration of trypsin and then as above.
posted by esnyder at 12:38 PM on May 18, 2009

You may also want to try a short pre-wash/incubation with trypsin (i.e. as if it was PBS) as well.
posted by Hutch at 1:55 PM on May 18, 2009

I normally detach cells using a similar method to scblackman:: pre-warmed versene/trypsin for 2 minutes at 37C, then bang the flask a few times and the cells detach. For tougher cells, I have used a higher trypsin concentrations and/or times of up to 10 minutes and bashed them quite hard without any obvious problems. I also do a quick pre-wash with versene before adding the versene/trypsin mix, I'm not sure how much difference that makes as I've never left it out.
posted by penguinliz at 2:21 PM on May 18, 2009

In following up with research on what is trypsin/EDTA, I ran across the following,

Trypsin:EDTA cell detachment process

We always use PBS w/o Ca & Mg to wash our cell cultures prior to the addition of trypsin/EDTA. I remember one day many years ago one of the lab scientists just couldn't get their cells to lift. Had about 8 flasks sitting in the incubator and the cells just wouldn't budge. Turned out we'd received a shipment of PBS WITH Ca & Mg and nobody had noticed! You definitely need to use a Ca free buffer to wash your cells with when using trypsin/EDTA.

posted by csw at 7:32 PM on May 18, 2009

I would also try increasing the trypsin concentration. I use 0.1% trypsin/EDTA (I call it 2x) with primary umbilical cord MSCs and fibroblasts, and also with carcinoma cells lines in the past with much more reliable results. I've also sometimes had trouble with late passage cells or cells that had been growing without being subcultured for a while and the 2x trypsin helps. I find that adding a fair amount of trypsin (~2ml for 75cm2 flask or 100mm plate) and leaving it on also works better than removing the excess and leaving a "thin film" of trypsin. Also be sure to put the plates in the incubator in a single layer on the shelf rather than stacked. I've had MSCs that laughed at 0.05% trypsin, but came right off in a few minutes with the 0.1% trypsin. It might also be worth using a fresh bottle or aliquot of trypsin.

Also, as csw alludes to, be sure you're washing the plates well. If the cells are done and you're just going to do something else with them (like get DNA/RNA, protein, etc.) you could just lyse the cells on the plate with whatever (e.g. Trizol.)
posted by sevenless at 9:01 PM on May 18, 2009

Without knowing the specifics of your trypsinizing procedure, I think that csw has the right idea. I was taught to wash the cells with PBS without divalent ions after removing the medium from the cells but before trypsinizing and the only time I've had problems with getting cells to come off was when we accidentally ordered PBS with Ca2+ & Mg2+. I don't, however, work with any really difficult cell lines (other than MEFs) but I've got coworkers and people in my department that do so I can ask around.
posted by The Bishop of Turkey at 6:37 AM on May 19, 2009

Response by poster: Problem solved: I had 0.25% trypsin / 1mM EDTA sent next-day, washed twice with PBS (Ca2+- & Mg2+-free, of course) beforehand, and saw the cells came off within 10 minutes. From now on I'll passage more frequently even if the cells aren't near confluence.

Thanks very much; these were all great suggestions.
posted by Mapes at 2:26 PM on May 19, 2009

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