Cannot get my babies to grow/proliferate - (Jul/20/2009 )

Hello everyone. I am having trouble culturing a certain type of cells. I've been a lab tech for about one year with previous labwork jobs in the past

I am growing mouse periodontal ligament stem cells that have been sorted with a fluorescent antibody to a protein we believe tells us if they are PDLSC's or not. The problem is they do not want to grow.

We extract teeth and they grow inside the teeth and then migrate outwards. They will flourish. After the first passage (using 0.25% Trypsin-EDTA for 3-4 minutes @ 37C), they stop dividing/growing. This has happened to me 3-4 times, so I feel as though it is the trypsinization that causes them to stop proliferating. They are not dying, as I have a culture flask about 6 weeks old with plenty of cells still in it, surviving, but not dividing. It was plated at around 60-70% confluency and it has increased in confluency.

I've tried growing the cells in many different mediums, with not much success so I'm hoping someone has a suggestion. I've talked about it many times with my PI and I don't think he knows what is going on either since his usual suggestion is for me to try and read up on the cells some more. For the most part, I've read all papers on Pubmed related to these dog-gone cells.
I've tried:
-DMEM Low Glu + P/S + Anti-mycotic + 10% FBS
-DMEM Low Glu + P/S + Anti-mycotic + 20% FBS
-DMEM Low Glu + P/S + Anti-mycotic + 10% FBS + 10% Mouse Serum (causes cells intially to grow much faster, but same problem at P1)
-AlphaMEM + P/S + Anti-mycotic + 10% FBS
-AlphaMEM + P/S + Anti-mycotic + 20% FBS
-AlphaMEM + P/S + Anti-mycotic + 10% FBS + 10% Mouse Serum (same as above with the MS)

I have not tried to exclude P/S or Anti-Mycotic as the conditions we must take the teeth are not sterile.
I use 0.25% Tryp-EDTA because these cells are extremely "sticky" and 0.05% Tryp-EDTA takes almost 20 minutes before they detach even with vigorous pipetting.
I have not tried to passage the cells with something other than trypsin, though I suppose that would be a good direction to go.

All suggestions welcome, and sorry for the long post, I just wanted to try and provide the reader with a good grasp of what I'm dealing with.

It sounds to me like your cells have become senescent. There is very little (actually nothing that I can think of) to reverse this unfortunately. You could try adding growth factors such as pituitary extract.

Also AFAIK, stem cells are typically grown in special stem cell medium.