Funny looking PC12-TrkB cells - (Dec/11/2006 )

This is such a great forum...it is the first time that I have posted, although I've been lurking for a while...

I'm having some odd problems with my PC12-TrkB transfected cells. I obtained the line from a frozen stock created by a long-since-gone grad student (2 years ago). There was only one vial - just enough to plate on T-75. I started the cells and they looked great. Nice disperse, smooth looking cells that responded to BDNF in culture. I passaged them 8 times, had to leave for a week, froze down half my stock and had a labmate take care of the other half. That left two vials of frozen stock and one T-75 flask growing.

When I got back, there was just a ton of junk in the plate. At first I thought they were infected, but I couldn't identify anything. Then I thought the collagen coating was coming off, but when I re-plated them, same problem. There was debris and cell bits everywhere. I kept the original culture going and started another from one of my frozen vials. Same thing. Now they've stopped growing so much, but they don't look good. The only way to describe it is that the inside of the cell looks "messy" - full of stuff, rough edges, etc. The PC12's I knew and loved from before were very smooth and clean looking with occasionall neurites and processes. They looked like big smooth blobs. Kind of like a water drop spreading out on something hydrophobic. These just look junky and full of stuff with little pieces floating everywhere..

I don't think they're infected with anything. They're not using up media very quickly, but they just don't look right to me. I don't have a ton of experience with cell culture, and so I'm not sure if these are safe to use (and don't know of anybody that could look at them here).

Any ideas?

I wondered if maybe they were just growing in clumps now and not adhering. Will PC12's grow in suspension? (I am growing them on 10ug/cm^2 collagen coated flasks).

I only have one frozen vial left and am very worried about losing this line. Thanks for any help!

I have the same problem with my cells though these are preadipocytes. They were fine earlier, but now even a freshly thawed out stock has plenty of debris and junk inside too. I have changed the media, serum, incubator and nothing seems to work. I don't know what to do.