What is wrong is -80? I have thawed cell lines that spent the better
part of a decade at -80 and they "woke up" just fine. The key is not the
freezing conditions, but they conditions that they were orginally frozen
The cells should be growing happily in log phase. Overgrown cells don't
thaw well. Also, what was the freezing medium? For long term storage,
use higher concentrations of FBS in the freezing media. Otherwise, they
won't last for a decade.
The hot tip to thawing is do the following...
Thaw the cell line in the morning. After thawing, spin out the DMSO and
resuspend the cells in culture media. At the end of the day, spin them
again to remove any residual DMSO that was in the cells. Keep them in a
small volume until they start growing well, then expand them. There may
be some contact needed to induce proper growing.
I agree with the 1:2 passages, but you should do it when you see debris
or dead cells. The dead or dying cells may secrete something that is
toxic to the living cells.
As for the media, try 10% FCS. I only use 5% when I am trying to slow
down cell growth.