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

I used to work with fibroblast type cultures while doing chromosome studies on some tumors. Our lab's protocol does involve chopping up the specimen and then processing it in collogenase for 30-1 hr in 37 C incubator before adding media. And we do try to add as little medium to the flask as we believed that having less medium would make it so that cells are more concentrated and thus more likely to start "sticking" to the culture surface. Additional growth supplements such fibroblast growth factors might also help.

Invitrogen table on growth factors for fibroblast

The table is for established cell lines of course, but as I recalled we used growth factors to help initiate our cultures.