Dear Research Community,
I am writing to seek insights and advice regarding an issue encountered with the revival of the MDAMB231 cell line.
Approximately one month ago, I preserved the MDAMB231 cell line utilizing a freezing medium consisting of 70% serum-free medium, 20% fetal bovine serum (FBS), and 10% DMSO. Prior to preservation, the cells exhibited robust health and vibality. The preserved cells were stored at -80°C for the duration of one month.
However, upon attempting to revive the cells, a concerning outcome emerged. Almost 90% percent of the revived cells were found to be non-viable.
I would greatly appreciate any insights?
Hello Asif Ahmad
I would consider three possibilities.
1. Did you carry out gradual freezing of cells when you prepared the cell stock?
The slow rate of freezing at an optimum cooling rate of -1 degree celsius /minute is recommended for mammalian cell lines. The rate of cooling controls the size of the ice crystals and the rate at which they are formed, both of which may affect cell recovery. I hope you have followed a gradual cooling rate of -1°C /minute. You may achieve this by storing the cryogenic vials in an isopropanol-containing cryo-freezing container such as Mr. Frosty and place them overnight in a -80°C freezer. This will allow the cells to freeze at approximately -1°C/minute, which is ideal for freezing most cell types. Also, you must freeze cells when they are in the logarithmic growth phase.
2. Did you carry out a quick thaw process?
You need to keep the cryovial in the water bath at 37 degree C until just the tiniest ice crystal is left in the cryovial. Two minutes would be enough for the cells to thaw with only tiniest ice crystals left in the cryovial. It is commonly assumed that cryopreserved cells must be thawed rapidly otherwise ice recrystallization events occur during warming which are detrimental to cell recovery. So, thaw the cells quickly in a 37°C water bath by gently swirling the vial and remove the vial when a small amount of ice remains. Do not vortex the cells.
After thawing the vial, immediately transfer the cells to a large volume of pre-warmed cell growth medium (~10 mL/1-mL aliquot of cells). You may either centrifuge the cell suspension and resuspend the cell pellet in fresh cell growth medium prior to placing cells in a culture dish or if you feel that the cells are too fragile to undergo centrifugation, you may plate the cells and change the growth media after the cells have attached (7-8 hours) or the next day.
3. Do you maintain a Temperature Log for the -80 Freezer?
This will help to record the temperature of the freezer on daily basis and corrective action may be taken at an appropriate time without any delay. This will prevent subjecting the cells to temperature fluctuations and thus maintain good cell viability on thawing.
Best.
@Malcolm Nobre thank you for your response. Yes, I preserved the cells via Mr.frosty at -80, and also carried out the quick thaw process. But I didn't maintain a temperature log for the -80 freezer
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