Is it possible to do a modification in cryopreservation protocol if -80 Degree storage is not available? We have 4 Deg C, -20 Deg C, -40 Deg C and LN storage.
Yes it is possible, but this is dependent on kind of cell line. I modificated cryoprevservation but only in the case of tumor cells. Cells are less viable after thawing. What kind of cells do you have?
Once I froze fibroblast without -80 degree and the cells don't survive.
Yeah Hyder, it is very much possible to do so. Slow freezing at these temperatures in descending order for half an hour each works for most of the sturdy cell lines.
I think it is possible, 4 C for 1 hr, then 2 hr for -20 C and then overnight for -40 C and lastly transfer it in LN storage.
I tried in the following way: -5 C 1 h, then 4 h for -20 C but I didn't have freezers for -40 C, and put in LN storage. But my cells didn't survive (it was MRC-5 cell line)
But cancer cell lines do not require early freezing.
I haven't tried it, but going straight into the -40C freezer ought to work followed 12-24 hrs later by LN. Best to use a buffering box meant for cell freezing if possible. If not available, then just use a small cardboard box (about 2" x 4" x 6") filled with crushed paper or styrofoam peanuts into which you should put your freezing ampoules filled with cells. The box slows the freezing rate to reduce ice crystal formation. Not necessary to go stepwise through 4C, -20C.
you can add more serum to freezing media 30% FBS then put cells in freezing media and cryotubes then move to box contain isopropanol to slower freezing then put in -20 for one day then move to -40 ... I think this will work specially to cell lines (MDCK, VERO, ....etc) but may be not for not primary cell cultures as (PHTBE cells) ...
We used to freeze in CO2 ice soaked in ethanol (this still lowers the temperature a few degrees extra), afterwards you go directly to LN, works fine.
Hi, thanks everyone for your excellent suggestions. I tried 30 % FBS, 10% DMSO in DMEM. Kept @ -20 Deg C for 2.5 hours, then transferred it to LN. Performed thawing after 72 hrs. Cells have revived and multiplying properly. Thanks again :)
There is no need to go 4 and -20 C. -40 C in a box full with conton or paper crush for 16-24 h then LN.
For next time, consider getting one of the isopropanol buffer containers - see "Mr Frosty" from Thermo Scientific. You can place your vials in this container and place the container directly at -40 C overnight. The next day, transfer vials to liquid nitrogent.
you have 4 Deg C, -20 Deg C, -40 Deg C and LN storage.
Add freezing mixture(DMSO+FBS) with cells.start with 1 hr 4'C,4-5 hrs -20'C,over night - 40'C then directly into LN.
Best Luck...
I use 5% DMSO and 95% FBS for as cocktails for cryomedium followed by the incubation at 4 deg C for half an hour, -20 deg C for 1 hour and then transferred to Liq Nitrogen.
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