I am going to culture the airway epithelial cell line A549.
After taking the vial out from liquid nitrogen, we have to incubate the vial in water bath at 37oC.
Why do we set up the temperature at 37oC but not higher or lower?
Hi Leo,
The cells should be thawed as rapidly as possible to minimize intracellular ice crystal growth during the warming process. That's why people generally use 37°C which is also the incubation temperature for most of the mammalian cells. Furthermore, the cell suspension should be diluted slowly after thawing as sudden dilution
can cause severe osmotic damage due to presence of DMSO and reduce the cell survival.
Most cell lines are derived from mammals (humans, monkeys, etc) whose body temperature is close to 37oC. Thus, frozen cells are thawed to this temperature and culture medium is immediately added to dilute compounds like DMSO, added as cryoprotectants, which could be toxic.
Thank Zaheer,
But my senior told that is not the reason :| I don't know her exact reason...
Hi Leo,I am pretty sure that all mammalian cell lines revive the best at 37oC and that because of the ambient body temperature maintained by most mammals...
Hi Charan,
is there no other reasons? For example the condition for DMSO or somewhat?
Hi Leo,
The cells should be thawed as rapidly as possible to minimize intracellular ice crystal growth during the warming process. That's why people generally use 37°C which is also the incubation temperature for most of the mammalian cells. Furthermore, the cell suspension should be diluted slowly after thawing as sudden dilution
can cause severe osmotic damage due to presence of DMSO and reduce the cell survival.
Thanks,
Do you think the temperature >37oC can make DMSO turn to be toxic for cells?
Hello Leo, as the others say, DMSO is toxic everytime for the cells when they are not frozen. So after thawing it is important to centrifuge the cells to excluded the DMSO also. Another point is that temperature over 37°C is generally not good for cells.
Maybe a hint for you. I also work with A549 cells and I make the observation that cells I froze slowly (-1 degree per minute) getting more damage then cells I froze faster. So in my experience I think A549 are very sensitive for DMSO.
37oC is optimum temperature for rapid thawing of cells, above 37o C increases cytotoxicity and decreses the revival rate of cell line.
We follow the protocol:
1 Rapid thawing of cell line at 37oC for 2 min
2 dilution 1:10 in culture media
3 Spin at 1250 rpm for 5 min
4 remove supermatnt and resuspend cells and seed in fresh T25 flask containing complete culture medium.
5 after overnight incubation change the medium to remove all dead cells (for adherent cells)
it gives better results
thanks
Hi Leo. DMSO can be toxic for some cell lines, but not due to the high temperature. The rapidity of thawing is critical, but temperature higher than 40oC does not fit to mammalian cells. If you thaw a DMSO-sensitive cells, you can look on thawing of icicle in the cryovial for the immidiate dilution of cell suspension. Here's it.
I suggest you take a look at this site with very detailed protocols and tips on the use of cell cultures
http://cellculture.altervista.org/
hope this will be useful even if long time is passed now!
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