Have I killed my cells? Doh! - (Nov/09/2008 )
Hi,
I recently attempted to freeze down some cells for the first time.
I needed to freeze down two different cell lines, & used 2X freezing media containing DMSO, FCS & DMEM.
As i had never done this before, I followed the protocol for one cell line first, & then the other. However after completing the first lot I left the cells at room temperature in the freezing media for approximately 30-45 min, while i repeated the protocol for the second cell line. I then put both sets of cells in the -80 freezer using Mr. Frosty.
It was only later that I realised DMSO is toxic to cells, and so I am wondering if it is likely that the first cell line, which was left for a while at RT in DMSO-containing media, are likely to have all been killed?
Thanks
I recently attempted to freeze down some cells for the first time.
I needed to freeze down two different cell lines, & used 2X freezing media containing DMSO, FCS & DMEM.
As i had never done this before, I followed the protocol for one cell line first, & then the other. However after completing the first lot I left the cells at room temperature in the freezing media for approximately 30-45 min, while i repeated the protocol for the second cell line. I then put both sets of cells in the -80 freezer using Mr. Frosty.
It was only later that I realised DMSO is toxic to cells, and so I am wondering if it is likely that the first cell line, which was left for a while at RT in DMSO-containing media, are likely to have all been killed?
Thanks
hi clarem
DMSO is toxic to cells at RT. am afraid that the first cell line will be badly affected after the exposure. however u can still give it a try and try reviving them. when u are reviving the cells, quickly thaw the cells and mix with media kept at RT. then u spin down to remove DMSO. u can repeat this step and then resuspend the pellet in very little volume. 3-4 ml. change the media daily and u can also make the media with 12 % FBS. this will help ur cells to revive better.
althugh the treatment was pretty harsh, but am hopeful u will b able to get the cells back.
if u are successful, then u grow them well and then make more frozen stocks.
if u are not very comfy with the freezing and all and ned more info, fel fre.
all the best
DMSO is toxic, it may not have affected your cells too badly but it is usually better to be safe than sorry when it comes to things that may affect your experiments.
When freezing cells it is best to keep them cold after addition of the freezing medium. My standard protocol for freezing involves pre-chilling the 2X freezing medium on ice, then re-suspend the cells in normal medium and cool on ice for 3-5 minutes and add the cold freezing medium drop by drop with mixing, then place tubes back on ice and into Mr Frosty and -80 as soon as possible.
You are better off getting all the cell lines to the chilling stage before adding freezing medium and then adding it to all of them at once, than you are doing them sequentially for the whole process.
Thank you both for your replies.
I was lucky that the first cell line which was left in the DMSO at room temperature was the least important of the two cell lines and so it doesn't matter too much if they are dead.
It's good to know though for future reference, I definitely won't be doing that again! 