Problem with Thawing (Freezing)
Posted 27 October 2010 - 12:05 PM
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!
Posted 27 October 2010 - 03:09 PM
Don't worry about the DMSO, it is being removed by the spin and resuspend steps you are undertaking.
Posted 27 October 2010 - 06:49 PM
Do the eye cells need a special medium additive?
Posted 28 October 2010 - 06:58 AM
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 :-)