Freezing and thawing of cells - (Sep/09/2004 )
we had also problems and know we thaw our cells more gentle, try it!
1. The cells from N2-tank into ice
2. some COLD medium into a falcon
3. some WARM medium in an other falcon
4. thaw the tube with your cells in warmer water then 37°C under gentle shaking, a little bit ice can believe in the tube, it will thaw until you are under the banch.
5. drop slowly the cold medium to your cells (2/3) more the solution was before
6. centrifuge at 300 g for 5 min at 4°C
7. discard the supernatant and resuspend your cells in the warm medium
8. now you can take them into culture
Thank you for your feedback! It adds to my knowledge.
I want to add other suggestion to your problem,at freezing as well as on thawing. Make pellete at 1000 rpm for 5min in round bottom tubes , discard supenatent,then add pre-cooled(1-2 deg.C) Freezing medium(DMSO 10%+FCS/FBS30%+maintenace media).Thawing ,take out Vial from liquid nitrogen,then wait for melting till small round ice ball remaining,then instantantly centrifuge it at 1000 rpm,then discard freezing medium,suspend cell in to media(supp.with 10% FCS).change media next day again.
Thank you for your suggestions!
If you are freezing you must be slow, if you are thawing you cant let them in the medium + DMSO or something else, so you have to be quick! Do not wait like harsh said! DMSO is zytotoxic!
I usually thaw cells in 60 deg for max. 1 min - wait till they detach from the cryo tube walls. then immediately place them in 15 ml falcon tube filled with about 13 ml of pbs. spin 500g for 5 min. usually works.
To inactivate FBS place in 55 degree C water bath for one hour.
I m doing cell culture from three year but recently i changed my protocol for thawing.Take cryovials from LN2 and just melt down till small roundish piece of ice is left ,put it into precooled centrifuge and centrifuge at 1200rpm for 5 min,after that decant the freezing media.Pour media and distribute in flasks,it working better for me.
I m also putting reason with it,DMSO will diffuse inside cells after temperature reaching at certain point(above 4 degree C.),so there is no toxicity if temp. is bellow 4.C tilldecanting of freezing media.
I dont think you had any problem in the method .Check the viability of the cells before freezing .
I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree.Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?
You might want to check the DMSO concentration that you use to make the cell stocks. If you are using 10% DMSO, try reducing it to 5%. If you are using 5% DMSO and 95% complete medium, try using 5% DMSO and 95% Serum.
While freezing cells, use IPA bath which helps in reducing the temperature gradually. Don't keep your frozen stock at -70 degree for too long, transfer it to LN2 after keeping cells at -70 degree for 24 hrs.
While thawing cells thaw them at 37 degree and dilute them with the medium as soon as the stock is thawed. You need not spin the cells, just dilute the cell suspension in complete medium so that DMSO concentration is less than 1% and seed the cells in tissue culture flask. When cells are attached replace the medium. It generally takes maximum of 2hrs for most of the healthy cells. Here I am assuming you are trying to revive anchorage dependent cells. If not you can spin the cells after 24hrs as long as your DMSO concentration in there is not more than 1%.
Hope it helps.
Wish you a good luck.