Hi, how do you store your long term stabilates in liquid nitrogen? Submerged or in vapour phase? Thanks in advance for any advice!
Dear Karolina,
It is important to slowly freeze the trypanosomes in isotonic buffered solution supplemented with 5 mM glucose and some DMSO to about -20C (no glycerol for trypanosomes). Once the ampules are frozen you can dump them in liquid nitrogen. Vapour or fluid phase N2 is not important. You may also freeze then slowly in a -70C deepfreezer, before transferring them to liq N2. If you have any more questions don't hesitate to contact me.
For the slow cool step in -80 we currently use CoolCell container, although they are rather expensive:
http://www.biocision.com/products/coolcell-cell-freezing-containers/
If you have any polystyrene containers which tubes used to come racked in, then they can be used in to sandwich the tubes for slow cool step. Freezer boxes are ok, they survive, but not sure if it affects longevity in storage, my sense is those aliquots have fewer life cells when defrosted.
We haven't confirmed if they recover better empirically, but anecdotally the length of time in -80C is also important, transfer them sooner to liquid nitrogen, don't leave them in there for weeks and months. I haven't read any advice on this, but within days, a week or two max. Other people's stocks in -80C stored for 1+ years tend to not recover well, stocks 5-10 years old do not grow at all.
We resuspend T. cruzi Epi 65% DMEM 20% HI-FBS1 5% DMSO for T. cruzi Epi, but you need to wash out DMSO when you defrost them. We freeze 1E7 cells/ml in 20% glycerol + 80% SDM-79 for procyclic T. brucei, and 5E6 cells/ml in 20% glycerol + 80% HMI-9 for bloodstream forms. Stocks I made several years ago still grow up. I add drugs same day for transgenic PF, but usually wait a day for BF to recover before adding them.
Hi Karolina,
In our lab, we mix trypanosomes stabilates with cryopreservant (20% glycerol in PSG) in ratio 1:1 then put in "Mr Frosty" for slow freezing for 24 hours then submerged in liquid nitrogen.
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