Cell Freezing protocol

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Guy Sovak
Guy Sovak's picture
Cell Freezing protocol

Hi all
Here is the protocol that I use routinely in the Lab I am using mainly two types of cell lines COS-7 and BHK.

Freezing Medium:
10% DMSO
20% FBS
Growing medium (in my case either DMEM or DMEM:F12 )

Trypsinize the cells
Spin down cells (1000rpm 2min)
Aspirate the medium
Add 2ml of freezing medium(per 1 10cm dish)
Pipette up and down the cells
Immediately transfer 1ml to cryo-tubes

Put in -80deg Celsius

JES's picture
 Anyone with a favorite cell

 Anyone with a favorite cell freezing protocol,
do you keep your freezing medium cold or room temp?
if cold, is there a max temp above which which you would start to worry after adding cells?
thanks for the help

Chin Fen Teo
Chin Fen Teo's picture
 I usually use the same

 I usually use the same freezing medium as gsovak's post- with 10% DMSO and 20% serum and process in a very similar manner.
As for the temperature, I usually use the medium at room temperature. However, immediately after aliquoting the cells, I will put them in a Mr. frosty (which has been sitting at -20 deg for awhile) and place the Mr. frosty at -80 deg right after I am done with the whole process...

JES's picture
 That helps! thanks.

 That helps! thanks.
Interesting!  A cold Frosty?
I find a variety of Mr. Frosty protocols that range from "must start at room temp"  to some using at 4ºC and it sounds like you are getting the Frosty to -20 before adding cells.
Do you let the frosty cool all the way down to -20 or are you just pre-chilling it to a less than room temp?
I was worried that -20 frosty might give a sort of soft-snap freeze than the -1C/min rate

Chin Fen Teo
Chin Fen Teo's picture
 Hi Jes, I usually put the Mr

 Hi Jes, I usually put the Mr. Frosty into the freezer the first thing I walk into the cell culture room before I start the trypsinization as such...  So it will be in there for like 10 to 15 min before the cells are in there... So, to be precise, it is not really at -20 degree... Sorry for the confusion and thanks for correcting me! Good Luck!  

heehawmcduff's picture
If you are worried about too

If you are worried about too quick a drop down to -20 just wrap your cryovial of cells in several layers of laboratory roll - it will cool slowly down to -20 or -80.

R Bishop
R Bishop's picture
I usually resuspend my cell

I usually resuspend my cell pellet after trypsinization in cold (ice bucket) 20% serum in my media, then leave them on ice for 5 min before adding an equal volume of 20% DMSO in media, leave that on ice for 5 min, then freeze the cells at -80 in between 2 -styrofoam racks that used to hold 15ml conical tubes. Works like a champ.
So for me the answer is ice cold media.

Guy Sovak
Guy Sovak's picture
As I noted I will add the

As I noted I will add the trypsinized cells to the cold freezing medium , then will transfer them immediately to -80.
Never had a problem with, Nih3T3, Cos-7 or BHK cells.
Important to remember the when thawing this procedure should be done quickly as the medium is toxic to the cells

The FFM's picture
Everyone seems to have their

Everyone seems to have their own variations on a core method that seems to work well enough for them.

Has anyone here actually done a controlled side by side study of different commercial cell freezing devices and compared it with some of these other  "home made" techniques in their lab.  Quantifying cell survival rates on thawing and starting the lines back up again for each technique?

If you already have the the data please do share it here.

PS - have routinely used a method like the gsovak one described in the first post, but in a 1 ml volume and placed the cryovial in a styrofoam box to go in the -80deg Celcius freezer to slow the freezing process.