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Cryopreservation (freezing cells) without using Freezing Container


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15 replies to this topic

#1 flashboy

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Posted 12 February 2002 - 06:21 AM

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?]

#2 nevermore1

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Posted 12 February 2002 - 08:36 PM

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%.

#3 BenJS

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Posted 13 February 2002 - 10:08 PM

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.

#4 bassman

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Posted 03 August 2004 - 06:34 AM

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

#5 ramakn

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Posted 04 August 2004 - 12:05 PM

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

#6 preeti

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Posted 06 August 2004 - 10:40 AM

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

#7 eric1turgeon

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Posted 10 August 2004 - 12:16 PM

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!!

#8 orexin2003

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Posted 23 October 2004 - 01:16 AM

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.

#9 Daniel5306

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Posted 16 December 2004 - 02:06 PM

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

#10 drB

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Posted 06 October 2009 - 12:14 PM

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.

#11 bachai

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Posted 06 October 2009 - 03:46 PM

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.

#12 lai55

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Posted 08 November 2010 - 06:20 PM

Thanks...


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#13 scifistudent

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Posted 14 March 2011 - 10:41 PM

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

#14 AntonAleraLabs

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Posted 08 April 2011 - 04:59 PM

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

#15 bolcitybol

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