thawed cells dying - (Jun/25/2007 )
Hi,
I had thawed HT29 from liquid nitrogen. the cells are not attaching to the surface after 3 days. after that i spin it down and transfer to another flask. but the cells still not growing and attaching. they seem have been dead. i think it is not the problem of the stock because the others are using the same stock also. what is probably be wrong?thank.
if the cells doesn't attach tye next day (if your cells will definately attach the next day)... they are probably dying.
maybe the person who freeze down the cells doesn't have a very good technique, or something wrong with the liquid N2 tank (forgot to refill the liquid N2?), or when you thaw the cells, you damage the cells accidentally.
quick thaw by immerse (half immerse) in 37C waterbath for not more then 5 min, when you see the half of the cells suspension thawed, quickly transfer it into a WARM media. spin down and discard the media with DMSO.
when you freeze down the cells, resuspend the cells in ice cold media and add DMSO (or any cryopreserve agent) dropwise. gently resuspend and transfer into vials. ice cold media may reduce the toxic effect of DMSO.
but i read from some articles saying that immediate spin down of cells after thaw will also damage the cells...is it that the HT29 is more resistant in this case? when i took the cells out fromt the cryovial and put into new media, the mix was already quite cloudy and yellowish...might this be the reason of the cells dying? i thought it might be contaminated before freezed or the cells number was too high...i had forgot to add in antibiotic when used the media..might bacteria contamination cause their death? i am already testing the media now...
maybe...
just use the media and plate it on LB agar and see....
good luck!
I don't spin down cells when I thaw them. I warm the vial in my hands and when I see that they are starting to thaw I pour them into warm medium. The following day I replace the medium. If the cells haven't attached by the next day, I spin them down and pipette into new medium. Are you sure the cells are dying? Have you stained them?
I had thawed HT29 from liquid nitrogen. the cells are not attaching to the surface after 3 days. after that i spin it down and transfer to another flask. but the cells still not growing and attaching. they seem have been dead. i think it is not the problem of the stock because the others are using the same stock also. what is probably be wrong?thank.
may be they were damaged during the freeze protocol; try to offer an excellent coating of the flask to get some attached
Dear HK,
I have initiating cells from frozen vials for 30 years now. I have been teaching cell culture for about 23 years and have ALWAYS centrifuged my cells (5 mins at 100g), both primary and cell lines. It has been my experience that by not removing the DMSO it is more damaging to the cells than centrifugation. DMSO is cytotoxic to all cells, 0.1% is the limit for most cell lines. However primaries are more sensitive and this % should be reduced to 0.01%.
So if your cells are frozen with a DMSO concentration of 10%, which is the norm, then you should be diluting your cell suspension/DMSO mix by a factor of 1:100 for cell lines, 1;1000 for primaries. The frozen vials again are normally 1-1.5 mls, then this is also a huge waste of media and very costly.
Also by having DMSO there for 24 hours, you will be SELECTING out cells that are more resistant to DMSO than others....this is also not a good idea.
Many regards
Rhombus