hello
when I preserve my cancer cell lines (MCF-7, HEPG-2, HCT-116) @-80 ْc for more than one month their appearance changes and after the third passage they died or contaminated how to overcome this problem in case of the absence of liquid Nitrogen
Dear Alaa Abdullah El-Sadieque what is your cryopreservation medium? usually we first freeze cells first at -20 for at least a day, and then move them to -80 since ultra freezing at -80 causes cancer cells to undergo severe changes to survive. Also, are you using antibiotics? If yes, then did you identify the contaminant? it could be mycoplasma.
Alaa Abdullah El-Sadieque There are a few steps you need to follow to cryopreserve:
These steps should maintain cell viability for a long period of time (I have cultured cells after 1 year from -80oC).
To reduce contamination during thawing, clean the outside of the cryovial with 70% ethanol in a biological saftey cabinet before opening.
Let us know if this advice helped - Good luck!
Dear Jack Hopkins
Prepared the freezing media that consist of (90% FBS and 10% DMSO)
then I Resuspend the cells (>=90% confluence) in1 ml of the freezing media and Put them at cryovial that following by placing them at -80
what is wrong can you help me
Alaa Abdullah El-Sadieque your freezing media should be 80% DMEM (or other cell line media), 10% FBS and 10% DMSO. Also, your confluency is far too high - your cells will not longer be in log growth phase and will not be as viable when thawed.
What is the number of cells per 1 ml and do you use a freezing container?
It is about 5*10^6 cells/ ml
I do not understand what the high confluency cause could you please explain in details
@jack
Dear @rehab
The freezing media consist of 10% DMSO 90% FBS
Alaa Abdullah El-Sadieque your cell concentration is far too high - reduce to between 1.0 x 106 to 2.0 x 106.
From the image attached, you can see the growth curve of cells in a closed environment. Ideally, you want your cells to be in the second stage (the log growth stage). Here they will be dividing rapidly and will continue to do so after thawing.
If you cells go past 80% confluency, they will begin to enter the third stage (stationary) and then the fourth stage (death). This is due to competition for space and resources. So make sure you are only freezing cells at about 60-80% confluency to ensure they are still dividing.
I hope this helps!
- Badges
- Science topic