Help: problem with MDCK cells - (Jun/14/2011 )

Hi, folks,

please help me figure out what's going on with my MDCK cells.

I'm growing MDCK cell in DMEM+10%FCS, and subculture them every other day by trypsinization (0.25%typsin-EDTA, 37C for ~6 min and then remove the trypsin and keep at 37C for another 6 min, hit and tap the flask to detach the cells). It worked well before. but now the cells become very weird. they grow over each other and aggregate, and some are weirdly shaped. they keep dying for several batches. so 2 weeks ago, I switched to MEM+1 mM L-Glutamine+10%FBS. they grew fine for 2 weeks, but were dead again this Monday.

I initially suspected they were contaminated by other virus, because they didn't look like infected by bacteria or mycoplasma. but it turned out to be negative for the other virus kept in our lab (containing GFP, so easy to check for contamination).

And the weirdest thing is that there are other guys in our lab growing the same cell using the same method, and their cells are fine. and all my cells are obtained from them which are fine before getting to my hands. the guy who taught me cell culture watched me doing it and said he can't find anything wrong with my method, but still my cells keep dying while others are fine. It has been torturing me for over 2 months and I can't figure out what's wrong.

I'm attaching photos of the cells. PLEASE PLEASE PLEASE help me! thanks!

The clumping together could be because you aren't resuspending them to a single cell suspension well enough before replating?

Also, are you sure that you are making up your media properly (I only ask because another grad student in my lab had all of her cells dying because she had added the wrong amount of a media component to her growth media).

leelee on Wed Jun 15 03:33:53 2011 said:

thanks a lot for you reply. I guess that the clumping together is due to new cells grow over old cells (those failed to dislodge from the previous passage), as you suggested. the cells are very hard to detach, so there are usually a couple of blocks of cells remain attached to the surface even though I keep them in trypsin for prolonged time. I usually flush them against the corner of flask ~10 times, sometimes even up to 20 times to separate them to single cells. I did ask the post-doc in our lab whether the old cells would cause problems, and he told me they wouldn't.

for the media, I suspected something might be wrong, so threw them and made new ones a couple times, but the cells are still dying. It has been the 10th batch that died! I've been working with MDCK cells for ~10 months, and they were fine before, but now they keep dying without noticeable reason. feel very depressed~