7 replies to this topic

[Nath]

Hi all,

While freezing suspended cells in liq. N2, the general protocol says to use 20% DMSO in medium. Does anyone here know the highest limit of using DMSO for freezing suspended cells like Jurkat?

Thanks.

[miBunny]

I froze Jurkats with 10% DMSO (final concentration).  20% seems rather excessive

[Stephan]

Why do you want to increase the DMSO?  The DMSO just prevents ice crystals from forming.  The less DMSO the better in my opinion.  I use 7,5% but most do use 10% DMSO in FBS.  At this concentration no ice crystals form so your cells "should" survive.   Remember to freeze them slowly!

[Nath]

Thanks for the replies. I am freezing cells in 20% DMSO, in a container with iso-propanol to drop temp quickly, before transferring to liq. N2. But while defrosting, I see cells broken and disintegrated for some reasons. I thought might be because of DMSO which was not enough to prevent ice-crystal formation which resulted in breaking cells. If this is not the case, what else should I suspect more ?

Thanks again.

Nath.

[lab rat]

Hi Nath,

The isopropanol will freeze the cells slowly (about -1C per hour). Thaw the vial quickly, under running hot water, and put the cells into cold incomplete medium asap. After you pellet the cells, then add the warm growth medium and seed the flask.

DMSO will prevent some enzymatic reactions from taking place, and can kill the cells. If you still have problems banking cells with your current medium, try a commercially-prepared freezing medium. I use Optifreeze.

Regards,

lab rat

[bob1]

lab rat, on Aug 26 2009, 02:50 PM, said:

Thaw the vial quickly, under running hot water, and put the cells into cold incomplete medium asap.

NO!
Thaw in the 37˚C waterbath and then transfer into warm medium, spin, resuspend etc.  If you thaw under hot water you are liable to kill your cells, and placing them into cold medium from hot induces heatshock (and coldshock) proteins that can kill the cells.

DMSO kills the cells by damaging the membrane - it dissolves the lipids.

20% DMSO is large, I use 10% DMSO, 10% FCS and 80% medium.

[miBunny]

Let me echo bob1 here...

NO!!!!

After thawing the cells (under room temperature water until just melted) put the cells on ice.  Transfer the cells to a 50 mL conical.  Add complete media (you need the 10% FBS to help buffer the cells because the cell membranes are damaged) slowly to the cells until you have 10 mLs.  Spin down gently.

DMSO is a cryopreservant because it enters the cell membranes and helps to stabilize them.  It also dehydrates the cells to decrease ice crystal formation within the cells.  And it decreases ice crystal formation outside of the cells.

The cell death you are seeing may be from how the cells were frozen.  The cells need to be very healthy and in the log phase of growth  (not overgrown) when frozen down.

[Stephan]

Agreed, you want to warm them up fast but nothing higher than 37C.  DMSO is toxic above 4C.  I think even if cells are frozen down 100% perfectly and thawed 100% perfectly, cell death is present in considerable numbers.