Freezing and thawing of cells - (Sep/09/2004 )
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?
I didn't see anything wrong with your thawing procedure. Are you sure the freezing procedure was right?
For making the liquid nitrogen stock,we usually pellet down cells at 1500rpm for 5 min. the remove the sup.
ater that resuspend the peleet in freezing mixture 1.5ml (FCS : DMSO :: 9:1).
then place the cryovial at -20 degree for 4-5 hrs then shift it to liq. N2.
If this procedure is wrong then what all modifications do i make? PLease suggest me some way.
the general rules for freezing and thawing cells is: freeze cells slowly but thaw cells quickly. It seems to me that you have not frozen your cells in a gradual manner. There are many ways for achieving gradual decrease in temperature. Check the section for cell preservation on this site and you will find protocols of different flavors for this purpose.
one thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.
Why do you want to pellet your cells after thawing. From your procedure you are using DMSO (1:10). DMSO will not give toxicity at low concentration
My suggestion is to add thawed cell suspn to 5-8 ml culture medium and incubate for a day then change medium. Initial pelleting could be avoided.
I never do a pelleting when I thaw cells (from -85 deg C). I never had a problem with low percentage in revial.
Good luck next time
try freezing cells in glycerol wid fcs than dmso
trust me this works
Preeti is right.
Most of the time I also use Glycerol instead of DMSO.
Glycerol with FCS in your culture medium will be excellent.
What is main difference in our procedure is that we centrifuge only at 1000rpm. I think that 1500rpm might be too much for some more sensitive cells. Moreover 1000 is more than enough. The cells during and after thawing have weaker membrane and in 1500 it might not hold.
try freeze the cells at -20 for 2-3 hours, then -80 overnight before placing then in liqN2