Our protocol for freezing cell lines is to supplement a single-cell suspension with 10% DMSO and slow-freeze overnight at -80oC in a commercial container (e.g. Mr Frosty). The advice is to transfer them to vapour phase liquid nitrogen ASAP. For logistical simplicity, some people continue to store their favourite lines in the freezer, accepting "some loss of viability" over time. I admit to having forgotten to transfer on a few occasions :-(
Does anyone have a more concrete data than "some loss" for the deterioration of frozen cells in the freezer compared to liquid nitrogen?
We have lots and lots of data on freezing B cells at multiple temperatures and in different freezing media. Not sure of the cells you are trying to freeze and then store for extended times, please indicate. Also, what is your criteria for "some loss"? i.e. viability, cell fuctionality etc.
David, many thanks for volunteering to share data! Most of my cell lines are epithelial (breast cancer) which are trypsinised into a single cell suspension for freezing. The trypsinisation does not cause a reduction in viability either immediately or after re-plating.
"some loss" is vaguely defined by viability upon recovery. For example, I tend to freeze the equivalent of a near-confluent T25 flask. When I thaw such an ampule into a T25 flask, a good freeze will be near-confluent while a bad freeze can result in 90% of the cells not adhering (on the few occasions I tested it, they were actually dead).
For the sake of others searching for similar information, I would be grateful if you could spare a moment to sum up the bottom lines you have learned for B cells within RG. If I could use more detailed information about any of them, I'll drop you a line.
Many thanks again,
Anna
I forgot my cryotubes in -80C for 5 days, then moved to liquid nitrogen. My cells (fibroblasts and granulosa cells) viability and attaching ability to the culture dish was diminished almost 40% and the cells became more bigger in size.
In my experience Anna its best to get breast cancer cell lines into the liquid nitrogen within 24 hours after freezing as this maximizes your chances of successful recovery. However I have left them for up to a week in the -80 and still recovered 80%.
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