Hi Lael,

You should be able to use a fairly standard method for starting primary fibroblasts. I wrote a detailed description of our method at RG a few years ago in the Questions asection. Look for it to get details. There are no enzymatic digestions, no specialized growth substrates, no cell type separations or centrifugations, or special growth media required.

We mince tissue biopsies into ~1 mm cubes using opposed sterile razor or scalpel blades, place the pieces into tissue culture grade plates without culture media, allow them to dry briefly (a few minutes) to promote adhesion, firmly press a dry sterile cover slip onto the biopsy pieces, add DMEM to the plate, and then culture for 10 days or so. The fibroblasts will grow out of the biopsy onto the coverslip and the culture plate. When they have grown out sufficiently, remove the cover slip, trypsinize the cover slips and the plate, and you’ve got primary Fb cultures. We used skin or other organs as the tissue source, but not intestine. However, the method ought to work on intestinal tissue too.

The method is effective due to the rapidity of Fb growth and division compared to other cell types, their strong adhesion to common charged substrates, and their resistance to damage by trypsin. They rapidly outgrow and out compete any other cell types present in the biopsy.

Good luck,

RW

Thanks! I also have experience with skin fibroblast culture (slightly modified from yours). However, I think intestine might be a little different....

Lael,

Yes, the isolation procedure may vary somewhat for intestinal fibroblasts as compared to Fb from skin or other tissues. Depending on the location in the GI tract (specify? ), such tissue samples may carry passenger bacteria, bacterial metabolites like LPS, or secretory material from mucosal cells including digestive enzymes. Any of these may interfere with establishing Fb cultures. Perhaps others on RG can offer recommendations more specific to intestine.

Best Regards,

RW