do we need to wait after adding in dmso? - (Jul/11/2007 )

hi,

i am trying to cryopreserve my cells. some articles mention that we need to mix dmso and cells and let it stay for at least 15 minutes for penetration before freezing it. some articles said no need. so does it really matters? my lab is short of -80 degree fridge. so i put the cells in -20 for 2 days..after that put into liquid nitrogen. is that ok?

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we mix the cells with DMSO containing media and then immediately place them on ice and freeze them in -80C . The cells are fine after thawing.

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we gradually freeze our cells, place the cells at 4 degree for a few hours after adding DMSO, and then a few hours at -20C and then we put them at -80C

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I've never incubated the cells with the DMSO/media before freezing and never had any problems. I've even resuspended the cells in the freezing media and thrown them directly into the liquid nitrogen. I may get a bit of cell death in the thawing but I always get enough viable cells. However, I highly recommend thawing a vial a few days after freezing to make sure your technique is good and the cells you have frozen are viable. This will also make sure you didn't contaminate them during the freezing (I've had this happen).

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the whole point of using the -80 rather than liq nitrogen is the slow freeze - you cells will be fine if you put them straight in the freezer (multiple freezers seems a bit of a faff, and straight in the liq nitrogen seems a bit risky - remember slow freeze, fast thaw)

dom

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Good post,

I do a similar thing:

10 minutes at 4oC
2hours at -20oC (in a polystyrene box with small puncture holes)
Overnight at -80oC ( in the same box)
Next day into liquid nitrogen
Week later take out a vial and do a viability test/sterility test.

The proper way is to buy a controlled rate freezer which takes the temperature down 1oC a minute, but they can cost upto £15,000. Primary cells are more susceptable to damage via freezing/DMSO than cell lines. If your cells have a high split ratio then you can get away with viabilty from frozen of lower than 90%.

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AFAIK the reason why cells have to be stored in liquid N2 (or -80) is that above -50 C there is a continuous "remodelling" of ice crystals, and it damages the cells. Therefore, if you store your cells at -20 C long-term, their viability will decrease a lot. I don't know whether 2 days is okay, but I would not risk it if I were you. I'd put them to -80 C right away in a cell-freezer container. If that is not available, a small cartboard box for eppendorf tubes will do the job (slow freeze). You can transfer them to liquid N2 the next day, so they dont occupy your -80 freezer for a long time.

Ooops. I just re-read your post. so you dont have -80 freezer at all? In that case you could try to reduce the length of -20 C step as much as possible. That means a few hours, max overnight. Hopefully, the rapid cool-down from -20 to N2 will not kill the cells. It doesnt make much sense to keep them at -20 C after they reached that temperature. Its worth a try if you dont have a -80 freezer.

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if you take a cryobox with isopropanol, it will take some time (I think ~1°C per min) if cells are cooled down from 37°C to -80°C; so the freezing program gives some time for the DMSO effect

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for bacteria (smaller size) it is OK to freeze them straight in liquid N2... but larger cell size, such as cell lines... freezing gradually is critical to maintain the cell viability...
and i normally resuspend my cells in ice cold media before adding DMSO. coz some people said freezing cold media can reduce toxicity of DMSO to cells.

Good Luck!!!

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I agree with the Bearer. we also use this isoporopanol to slowly cool our cells down to -80°C. in that case you don´t have to care about anything. it´s cool!

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