What type of cells are you using and what did you freeze them in? Hopefully you remembered to add some cryoprotectant (e.g. DMSO, glycerol) to the media when freezing down your cells. If you didn't, its likely they're all dead from freezing process. Also, if you describe the process you're using to grow your cells again, it'll help us to be able to give a clearer answer about where things might be going awry.

What type of cells are you using and what did you freeze them in? Hopefully you remembered to add some cryoprotectant (e.g. DMSO, glycerol) to the media when freezing down your cells. If you didn't, its likely they're all dead from freezing process. Also, if you describe the process you're using to grow your cells again, it'll help us to be able to give a clearer answer about where things might be going awry.

Hi, in my experience if you left cells (in my case muscular cell lines) for several month at -80°C they suffered a lot and when you thawed them you have an half dead, respect the total number frozen. If you have only this aliquotes I would try to thawn, as usual, very quickly, at 37°C in a water bath and seed them in small space and volume. For example if you normally put your cells in 10mm petri, try a 6mm petri. For the medium, as Philip asked you, you can write some information about your cells but I would suggest to add more serum, for example insted of 10% try 15%. I only want to advice you to pay attention with these cells even if you can "save" them. For example in my experience, with muscular cell lines, after a suffered period, these cells have problem with differentiation, so you have to be sure that your cells can be used for your experiments.

When you're thawing weak cells I'd say the most important factors would be protecting the cells from DMSO damage (thaw into cold media, don't let the cells spend too long thawing in the water-bath), reducing mechanical damage to the cells (pipette very gently), and increasing the concentration of the cells in your culture vessel after thawing them (thaw into a 6/12/24 well plate instead of a T25). 

I've heard some people forgo spinning down their thawed cells, instead increasing the amount of media in the culture vessel to dilute the DMSO, then changing the media later/the following day when the cells are adhered. I suppose it depends on what's killing your cells as to how best you can proceed. Good luck!

Edit: As Valeria said above, if your cells *do* survive, keep an eye on them for strangeness. If 95% of the cells you froze down die and you manage to get the other 5% going again, you might be forcing some kind of clonal selection or the stress of that might have changed them in some way.

thanks philip and veleria..

my cells are transfected MEFs.and i had selection by abtibiotic after transfection.and i expanded them an freezed by 10% DMSO , 40% DMEM, 50% FBS.

and when i wanted to thow them  i gelatinized one well of a 6well plate and used that.i know staying in -70 (as i know our freezer had problem in coolong s.th!) for along time kiilled my cells.in your opinion thowing them whiout pelleting can help me ?

Spinning down your cells in your normal way (not too fast) is probably good rather than bad - if they are 95% or 99% dead then some of the dead cells won't pellet and after removing the medium and resuspending, you may see your live cells (if any) better after plating.

One hint is to dilute out the DMSO slowly. This can greatly increase viability, as 10% DMSO is highly hypertonic (over 5 x isotonic) and cells can burst by osmosis if you dilute quickly.  Give it time to diffuse across the cell membrane.  With a healthy stock you can get nearly 100% viability this way.

So you thaw the vial quickly by dipping carefully in the 37C waterbath (but don't wet the screw thread).  Pipette your thawed cell suspension (1 ml probably?) gently to detach cells from the bottom of the vial, and transfer it into a 20-ml centrifuge tube.  Now take 1 ml of growth medium and slowly add a drop at a time with swirling, over about 30 sec.  Now add another 1 ml over about 10 sec, and then another 18 ml over 30 sec. 

Now centrifuge and (if your cells are mostly dead) resuspend the pellet carefully in a small volume - could even be 0.5 ml and place in a well of a 24-well plate.  Or 2 ml into a 3-cm well or dish as you say.  Leave them alone and don't look till next day.  If they are not attached by 24 hrs, they are probably dead, but maybe worth keeping to see if a few colonies grow.

Good luck!

Hi, you got a very exaustive answer to your question above! 

I think that your problems were caused by the several month that your cells were left in the freezer, I have a -80°C that have problem to reached this temperature and it was always around -60°C. if I thawn my cells wrapped in simple sheet paper, without the help of freezing container and used them after few weeks I got good result, but if I thawn them after 1or 2  months I find a lot of dead cells. As Dr Bennet said try and see if you see colonies grow because I think your cells are stable clone generated after transfection and selection so it is worth the time to save them.

thanks a lot dorothy .. i will try your suggested method.i hope to have my cells..