15 replies to this topic

[flashboy]

I have been having trouble with viability of my BRIN-BD11 rat beta-cells recently, and would like to know if anyone has a good method for freezing down cells that doesn't involve paying £50 for a Mr Freeze cryo tub [any budding home improvers out there who can suggest a way to build one?]

[nevermore1]

I have had very good success in freezing cells by simply putting the Nunc vials (or whatever you're using) into a
stryofoam box in a -80 C freezer for 24 hours then transferring directly to liquid nitrogen storage.  The box should already be in the freezer before you start.  I've done this a couple of hundred times and always had viabilities of at least 70%.

[BenJS]

Agree with the last post. Even simpler, we used to place the tubes of cells over a canister of liquid nitrogen for an hour or so first, then to -80 for 5 hours and then to the storage canister.

[bassman]

i agree with nevermore. used that method tenths of times.

[ramakn]

The technique mentioned really works well also take care that you have 950 of your cell suspension and 50 of sterile DMSO

[preeti]

i frz cells regularly by putting them in cryovials(at 4 deg C), transfer to-20 deg C(1hr),shift to -80 deg C for few days & transfer to liq nitrogen
preeti

[eric1turgeon]

The trick is not to freeze your cells too quickly. By putting them in a styrofoam box in the -80 freezer overnight, it allows your cells to freeze down slowly. Works great!! Liquid nitrogen next and you're in business!!

[orexin2003]

I agree with preethi, i find freezing the cells at 4 for few hours, then at -20 for overnight then at -70 for week or so then transfetring to liquid nitrogen results in better stability of cell membrane, by following this method one would prevent the cells from undergoing cold shock which can result in necrosis of cells.

[Daniel5306]

My method is more simple. I directly put the tube into -80oC. Then keep there for years. The viability of the cells usually are more than 80%. I used this method for at least five kinds of different mammalian cells, it works very well. I have to mentioned that the medium what I used to store the cell were 90% of FBS and 10% of DMSO. Good luck.

Daniel

[drB]

I agree with Daniel.  Although no two cell types will react the same to cryopreservation, we also find that the 10% DMSO in FBS works much better than 10% DMSO in DMEM in almost all cell types.

[bachai]

drB, on Oct 7 2009, 06:44 AM, said:

I agree with Daniel.  Although no two cell types will react the same to cryopreservation, we also find that the 10% DMSO in FBS works much better than 10% DMSO in DMEM in almost all cell types.

I agree that the final 10%DMSO+90%FBS seems to be good for most of cell types.
To be more gentle, don't rush to mix cells with DMSO. Resuspend them first in 100%FBS and let them calm down on ice for 15-30min while you are labeling cryovials etc. Then add dropwise equal volume of 20%DMSO+80%FBS, mix well, aliquot into cryovals, and freeze them either in the expensive Mr.Frosty, or a cost-nothing foam box.

[lai55]

Thanks...


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[scifistudent]

Hi,

i agree with Nevermore. I worked on HepG2 cells and these cells were stored with 60% FCS + 30% DMEM + 10 % DMSO. We use cryovial from nunc and we first keep them in -20 oC (2 hr), followed by -80 oC (Overnight) and next morning we store the  vial in Liquid N2 cylinder.

THis works well.

Good Luck

[AntonAleraLabs]

I would second the opinions of using 90% FBS with 10% DMSO and keeping cells at -80C all the time even for long-term storage.


Anton
Principal Consultant, Molecular and Cell Biology

Phone: (518) 334-0922
E-mail: antonb@aleralabs.com
Web: www.aleralabs.com

Alera Labs
P.O. Box 13019
Research Triangle Park, NC 27709

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