Establishment of primary fibroblast culture - (Jan/24/2007 )

I have just started a new project where I need to establish primary fibroblast cultures, first on volunteers and then once it is working then on patient samples (around 20 - 30). I will probably only have one sample to work from for each so I need to get it going well ASAP!

I took my first volunteer samples 3 weeks ago and have seen no growth. They have been in DMEM with 20% FBS (seems a lot to me but that is what I was told to use), and I have tried both letting the fibroblasts migrate out of the 2mm skin biopsy and also with another couple of samples, tried chopping them up with a sterile scalpel and then leaving them to grow. None of the 5 samples I have have shown any sign of growth in 3 weeks.

My next move is to try a collagenase method I received from someone else, and also to try placing a sterile coverslip over the top of the cells as this apparently helps stick them to the culture dish and also creates a microenvironment where they can grow better.

Does anyone with experience of establishing fibroblast cultures have any suggestions or handy hints?? Thanks, anything at all would be much appreciated

well i don't know how waas established the IMR90 fibroblast culture, but in our lab, they are more fine in 10% serum.
Moreover the DMEM we use don't contain glutamine. We add half of normal quantity as the glutamine enhances urea formation in the medium.

I am also involved in a new project, where I need to establish primary fibroblast cultures from adult mouse skin. I have tried mincing, nonmincing, trypsin, no trypsin, and also have a collagenase protocol which I have not yet tried. I would be interested to know how your cultures turned out, as I will be taking biopsies again very soon.
Thanks in advance,
LC