Problem with Thawing (Freezing) - (Oct/27/2010 )

I'm having a problem when thawing out cells from liquid nitrogen. I am trying to thaw 3 cell lines: A549 (lung cancer), RG2 (rat glioma) and human eye cells. TO start with none of the cells were frozen by me. The eye cells were frozen by one of the immunology doctors. Likewise the RG-2 cells were frozen by one of the pharmacists, and the A549's were frozen by a grad student who has since graduated and is now long gone. When I thawed the eye cells out, not a single eye cell survived, a fair amount of the RG-2 cells survive, and only a handful of the A549 cells survived. I'm concerned about the A549 cells, since I only have 1 more vial of these cells, the others I have about a dozen.

The protocol for thawing that I was given to follow went as follows:

Keep the cryovial on ice, and quickly add 500uL of warm media to the frozen cells. Pipette up and down a few times and then transfer 1000uL of the contents to a 14mL centrifuge tube containing 10mL of warm media. Repeat this process until the contents of the cryovial are empty. Then spin the tube for 5 mins at 1.5g. Aspirate the supernautant, and resuspend the pellet of cells in 1000uL of fresh media. Transfer this media into a culture flask containing 20mL of warm media.

I had the tube spinning in a little under 3 minutes, so I would venture a guess that I satisfied the "quick" criteria. This is the same speed/lenght that we use when culturing cells, and it's been working without incident. And yet somehow the survival rates for the eye and A549 cells were none and a few dozen, respectively. This protocol worked without any real problems for the RG-2 cells.

The freeze media contains DMSO, but how much I don't know exactly.

To get to the point, I've got a handful of A549 cells being cultured at the moment, and 1 more vial still in the nitrogen. I would like to start doing cell uptake experiments as well as MTT's next week, so anything you much more experienced scientists can tell me to help more cells survive thawing (assuming the freezing procedure went properly) would be greatly appreciated. Thank you!

The usual rule for freezing and thawing is "freeze slowly, thaw fast" so as to prevent formation of ice crystals that will disrupt the cell membranes. Try thawing the cells in the palm of your hand or in a 37 deg C waterbath until the ice is gone, then treat them as you have described, which all appears fine to me.

Don't worry about the DMSO, it is being removed by the spin and resuspend steps you are undertaking.

We thawed ours under running hot water until the ice was the size of a pea, then transferred the whole volume to 10 or so mls of prewarmed medium. Spin at RT, harvest cells, transfer to flask.

Do the eye cells need a special medium additive?

The eye cells do require some additives to the media, exactly what and how much I can't remember off the top of my head. The media I used when attempting to thaw them did contain all of the required additives.

Thank you for confirming that the DMSO is removed in the spin. I changed the media 18-22 hours later just to be absolutely sure that all the DMSO had been removed. It helps me sleep at night :-)