Especially tenacious cells -- what to do? - (Feb/22/2010 )
Hello, all!
Your collective expertise is needed. I'm growing cultures of a transformed murine middle ear epithelial cell line. They are, in most respects, a dream to work with. They grow quickly, they're very hardy, etc. The only problem that comes about is that they are nearly IMPOSSIBLE to trypsinize easily. I wash the attached cells at least three times with PBS to get rid of any possible lingering traces of serum, but they absolutely bluntly refuse to detach from the flask with anything less than a ten-minute trypsinization. The problem this poses, of course, is that when they finally do detach, they clump like nobody's business, and it's very very difficult to get them to resuspend in fresh media, and it's also seriously decreasing my cell viability.
Any suggestions? Less clumpy detachment methods? More trypsin? Wash the cells more? Some variety of cell-detachment dance? I'm willing to give anything and everything a shot. We're doing SILAC soon, and with the money we're investing in it, I want to make sure everything goes as smoothly as possible.
Thanks in advance for your input.
(Reposted from "General Laboratory Techniques" because I'm an idiot.)
Do you use trypsin-EDTA? If so, try just plain trypsin, it should be less damaging, though the cells might clumps a little more. Also have a look at the ATCC website to see what they suggest if they have your cells in stock.
I don't know if it's possible for my cells to get any clumpier than they already do. 
I suppose my biggest concern is that I don't want to treat them too roughly. They're pretty sturdy, but it's clear that the substantial amount of pipetting that they have to go through to resuspend thoroughly has a negative impact. Would they clump more but be easier to resuspend if I used plain trypsin (yes, I am using trypsin-EDTA currently)? If so, why is that? Also, if I'm washing two or three times with PBS, will that get rid of sufficient Ca++ that the EDTA won't be necessary?
bob1 on Feb 22 2010, 07:34 PM said:
clickpopclick,
I've nver had to do this myself, but some protocols call for passing clumpy cells through a syringe with needle to disipate the clumps. I may be able to dig up a protocol if you are unfamiliar with this technique.
Hope this helps.
Kevin
clickpopclick on Feb 24 2010, 07:46 AM said:
I suppose my biggest concern is that I don't want to treat them too roughly. They're pretty sturdy, but it's clear that the substantial amount of pipetting that they have to go through to resuspend thoroughly has a negative impact. Would they clump more but be easier to resuspend if I used plain trypsin (yes, I am using trypsin-EDTA currently)? If so, why is that? Also, if I'm washing two or three times with PBS, will that get rid of sufficient Ca++ that the EDTA won't be necessary?
They should come off the plates a lot more easily with just plain trypsin, but it is a bit more damaging. Washing Ca2+ and Mg2+ free PBS (Dulbecco's-PBS) should be fine.
KevinK on Feb 24 2010, 05:30 PM said:
I've nver had to do this myself, but some protocols call for passing clumpy cells through a syringe with needle to disipate the clumps. I may be able to dig up a protocol if you are unfamiliar with this technique.
Hope this helps.
Kevin
That's not a bad idea. If you've got something handy or easily findable, I'd like to see that. I'll look around on my own and see what I can turn up.
bob1 on Feb 24 2010, 06:17 PM said:
clickpopclick on Feb 24 2010, 07:46 AM said:
I suppose my biggest concern is that I don't want to treat them too roughly. They're pretty sturdy, but it's clear that the substantial amount of pipetting that they have to go through to resuspend thoroughly has a negative impact. Would they clump more but be easier to resuspend if I used plain trypsin (yes, I am using trypsin-EDTA currently)? If so, why is that? Also, if I'm washing two or three times with PBS, will that get rid of sufficient Ca++ that the EDTA won't be necessary?
They should come off the plates a lot more easily with just plain trypsin, but it is a bit more damaging. Washing Ca2+ and Mg2+ free PBS (Dulbecco's-PBS) should be fine.
And the additional potential damage won't interfere with SILAC, you don't think? Or will it all end up balancing out if they come off of the flask more easily/less traumatically than with the trypsin-EDTA?
(Can you tell I'm fairly new here and scared of screwing up expensive things?
) Do you leave them at RT while trypsinizing? Sometimes putting the cells back into the incubator helps.
lab rat on Feb 25 2010, 11:23 AM said:
No, I pretty much have to put them back in the incubator (our lab in general, and the TC room, in particular, is frigid). I'm wary of warming them up any more than the 33 degrees I grow them at, though, because they differentiate at 37, which I'm avoiding for the time being.
clickpopclick,
The methods Ihave found use a 21 or 22 gauge needle on a 5cc syringe. Simply draw the cell suspension through it 5+ times to disperse the clumps. Again, I have no experience with this technique or how harsh this will be on the cells, but it would be easy enough to try.
Kevin
KevinK on Feb 26 2010, 09:45 AM said:
The methods Ihave found use a 21 or 22 gauge needle on a 5cc syringe. Simply draw the cell suspension through it 5+ times to disperse the clumps. Again, I have no experience with this technique or how harsh this will be on the cells, but it would be easy enough to try.
Kevin
Hmm. I'll try this when I passage my cells on Monday -- not sure how different it is from repeatedly pipetting, though I admit that I usually have to pipet more than five times to get everything broken up. I've also got a fair number to get through (I've got ten T75 flasks at the moment). I'll see how it goes!