One problem could be that they were kept for 3 months in -80ºC, although I would expect cells started dying from the beginning. 

These cells are IL2 depended and although they should produce ti they may not produce enough. Avoid diluting them too much. If they are diluted the IL2 they produce may not reach the concentration necessary. You could try rescuing a small alicuot of cells with recombinant human IL2 tosee If they improve/grow/stop dying.

Good luck

The cells needed to be moved from -80 to liquid nitrogen. I echo what was said above, these are probably shot. And yes, they HAVE to have IL-2. If they were frozen in freeze media, make sure you wash once when you thaw before giving them warm fresh media with 4% IL-2.

Thanks for your comments. I have started adding IL-2 but not 4% and I have not seen any progress in their proliferation yet. Does it matter if I have not used horse serum and I have used 20% FBS instead? 

I am affraid that congelation at minus 80°C is not sufficient for the cells and that they should be kept frozen in liquid nitrogen

I know exactly what to do, and I guarantee 100% it will work!

Call ATCC, and tell them.

They will probably send you out a new stock. by the do everything exactly how the atcc sheet says.

At minimum this will eliminate the 3 months at -80 being the problem. I would also be suspect of you IL-2, how old is it, do you aliquot it when you first open it? the entire vial shouldn't be unthawed everytime--that's a somewhat common mistakes I've seen before.

Also, contamination could be the issue, maybe with mycoplasma or something that isn't obvious.

The biggest thing with unthawing cells is to do it FAST, like under a minute fast (from the tank to the 37degree incubator. Also, prewarm the dish (with medium) you'll be using and the conical tube (with medium).

I know ATCC's sheet for L929 cells says, it should be no more than 3-5 min.

Also, you need to google a picture of what the cells are suppose to look like, and LOOK AT THEM CONSTANTLY.

The best advice I ever got with tissue culture was "look at cells". this really amounts to getting to know them.

what do they look like when they are hungry, or sick, or mutated? you should know that. What do they look like when they are freshly plated or at steady state, or in a monolayer? What's the difference between plating them on a petri dish or a tissue culture plate?

I emphasis this because you don't know when they went wrong. Maybe they were sick all along or maybe they got contaminated at day 6 and died the next day. To start you could do a viability assay after you first unthaw them.

But look at them, 4-5 times a day at least

Thank you for sharing your experiences with me. It is surprising to me that cells are growing in a slowly rate after they are thawed and I know exactly how they should look like. They should look like a grapes as ATCC said in the data sheet. I have been checking on them constantly and I observed that the cells are not forming any grape shape few days after thawing and they are scattered individually in the plate. About the IL2, I use the aliquots and not directly from the original vial.

What's the est. doubling time of cells?

The est. is about 2-3 days if they grow well.

Did you solve the problem? I am now facing the same problem with you. can you tell me how to do if you know the answer.

thank you,

lewis