Thawing cells - (Oct/01/2014 )

Hi guys,

My company recently moved to a new building, where procedures are somewhat different. Our cell stocks are now located in the basement and my lab is on the 4th floor. As the cells are stored in nitrogen, company rules obligate us to transport the cells via a route that takes about 5 minutes to get back to the lab.

For convenience, I transported my PC12 cell aliquots in buckets of watery ice. I assumed that the cells would suffer minimally from the transport as it's just a short trip. However, I noticed that when I take the cryovials from the nitrogen stock, the medium in which the cells are frozen is yellow, and upon arrival at the lab the medium has already turned pinkish-purple. This is a pretty big difference.

The last time I transported my cells like this, the cell survival rate was pretty low. The time I started up new cells before the last time, I still had some aliquots in the -80 freezer and using these vials, the survival rate was much higher. The trip from the -80 freezer to the culture lab is not even a minute, and I put the cells on ice like I do when getting my samples from the nitrogen stock in the basement.

I was wondering: as the cells are frozen in culture medium (10% FBS, 1% pen/strep) + 10% DMSO, could the 5 minute walk with my cells on ice from the basement to the lab already cause the cells to die that rapidly?

The color change indicates that the medium is thawing, so your cells could potentially be killed or damaged during the trip. If you have a Mr frosty container used for freezing cells in the -80, then you could freeze that at -80 and then use it to transport the cells to your lab.

Could be that the cells are dying because they you're thawing them to slowly.  Cells don't like to be in liquid DMSO for too long so a good rule of thumb to follow is to thaw your cells as quickly as possible.   Do you have dry ice at your facility?  The easiest fix would be to fill up a ice bucket with dry ice and transport the cells on dry ice.   When you get your cells back to the lab, take them off of dry ice and transfer immediately to a 37 deg water bath.  Once they're thawed add them to 15 mls prewarmed media and spin down then decant to get rid of the DMSO.   Media + 10% FBS + 10% DMSO should work but you could also try freezing with 90% FBS + 10% DMSO, or try a serum free freezing medium like Wako Bam-banker if you continue to have problems.  

Another thing to consider is it might be how you're freezing the cells.  In contrast to the rule for thawing cells, you should try to freeze cells as slowly as possible. Sometimes transfering them from room temperature straight to liquid nitrogen can lead to decreased viability after a thaw.   In our lab we usually put the cells in -80 inside a styrofoam box to insulate them overnight and then transfer them to the liquid nitrogen the next day.