I am trying to establish a primary culture of fibroblasts from mice. I was following a protocol previously used in our lab that isolates them from lungs, and I recently tried another protocol that isolates fibroblasts from ear clips. No matter what I do, no matter which reagents I use, I cannot get fibroblasts to grow (and these are protocols that should just work with minimal effort!).
I have been removing the tissue (lung or ear clip), rinsing in PBS, mincing with a razor, treating with trypsin (I've tried 0.05% for 1 hr and 0.25% for 10 min), rinsing with DMEM (+15% FBS +1% antibiotic) and then plating. I also tried an additional 1hr, 3hr, and 5hr 0.3% collagenase digestion on the lung tissue. With the lungs, I have a goopy mess with bits of tissue and over time just dead cellular debris at the bottom, and with the ear clips I am getting nothing but floating bits of tissue in media. Bacterial contamination is not a concern.
Anyone have any ideas why I cannot get fibroblasts to grow? I do not think I am over-digesting the tissue, and another individual followed the same protocol and it did not work for her either. Does anyone else get the goop (reminds me of cellular membrane debris, but I still have lots of bits of tissue)? Do I need to supplement my media with anything extra? Sing the cells a song, read them a book?
This is supposed to be very easy, and it's holding up several months of planned experiments until I can get these cells to grow.
Sincerely, Fibroblast Hater (Caitlin)