Hi everybody. I was thawing my cells which were stored in liquid nitrogen. I have never had any problem with freezing or thawing the cells for 5 years. But now there is problem with some of my samples. Cells are not attaching the surface (only very few of the cells) and it seems that most of the cells are dead. I was searching for some reasons why this situation could happen and the problematic samples were stored in liquid nitrogen directly in the liquid phase. The other samples, which were nice proliferating, were stored in vapor phase. Do you think that there could be some problem of liquid nitrogen itself or it might be some cross-contamination? Can only liquid nitrogen destroy the cells like this? Please if you have any suggestions I would be very thankful for them.
Storing cells in the liquid phase is an issue of safety, (the vials might burst when warmed), and a potential issue of contamination. Primarily it is a safety concern. The contamination I worry about is viruses from other samples somehow getting into the liquid. Even this is very very unlikely to happen. Any potential chemical in the liquid is also very unlikely to be the cause of what you describe. Almost anything that gets into the liquid will turn to a solid. I suspect that the cells in the liquid phase were not handled well at some point. Frozen too fast, frozen too slowly, allowed to warm above -90C, or some such.
I agree with Dennis, most cryovials are rated for vapor storage and can burst or crack if thawed too quickly. it is possible that the cells in liquid storage froze too quickly, or were moved to storage before they were at the appropriate temperature. Always freeze cells using a freezing vessel that will control the temperature to drop about 1 degree a minute in a -80 freezer. I use a stratacooler that is primed to 4 degrees after vials are added I move the stratacooler to the -80 overnight and then move the vials to liquid nitrogen. I use the following freezing media (10% DMSO and 90% FBS). I add 0.5 ml freezing media to the vials and then add cells in 0.5 ml growth media to dilute the DMSO to 5%.
what kind of cells. we use semen cryopreservation for long time using liquid nitrogen, but with cryoprotetor (glycerol), and a protocol to control temperature to drop 1 degree a minute in N vapor before put in a liquid N
Thank you all for your suggestions. Finally we tried to thaw another samples from liquid phase of nitrogen. After thawing and culturing the cells for 3 days it seems that many of them finally started to attach the surface (normally it takes about 6 hours or little more, but 3 days its too long). Cells are looking stressed and almost like going to senescence. Im very curious about this situation, because as I mentioned, I have never had any problem with cells before. Could it be caused by viruses? Im working with primary porcine fibroblasts. Our freezing media is ok, cause other cells are nice. Freezing protocol is also ok, we use freezing blocks (isopropanol) to allow freeze the cells 1°C per minute (in -80°C) and then I put them to Liquid nitrogen. And all samples were handling the same. Only now I have this problem.
It could be caused by a leak in the seal of the cryovial gasket. Also, liquid phase is much closer to the -180oC range while the gas phase is typically around -150 or -160oC, we have observed that the liquid phase LN2 can put greater stress on cells that haven't taken to freezing well. Also with previous experience, what passage are your cells comming out of thaw? Too high a passage or constant freezing/thawing events can put undue stress on the cells and reduce their viability.
I advise the gas phase of LN2 for long-term storage. Liquid can leach into external or internal threads on vials and cause contamination/explosion risk at thaw.
i have been using liquid phase one for many years without any problem.
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