I am working on isolating human liver fibroblasts. Does anyone have experience doing this and/or suggestions for doing this? So far I have tried culturing stellates and also mincing up liver tissue.
I never have attempted to isolate fibroblasts from liver tissue, but one general method for fibroblast isolation is to cut the tissue into very small pieces, place them on the bottom of a dry culture dish, overlay them with a coverslip, wick media under the slip with capillary action, and then gently add more media to the dish.
Wait about a week and see if you have fibroblasts growing around the periphery of the tissue pieces. When you have a sufficient quantity of cells, remove the coverslip and trypsinize the fibroblasts.
Hi Casey, I do not know if this helps, but I have experience in isolating fibroblasts from whole chicken embryos.
In a TC hood, cut up the tissues into small pieces, put into a 50 ml conical tube with 40 ml of 10X trypsin/EDTA with pen/strep and a stir bar. Stir the tissue for 20-30min at RT.
Separate tissue fragments with a low speed spin (~300 X G for 2 min). Transfer the sup to a new 50 ml conical and do a hard spin (~3000 X G for 5 min). Bring the pellet up in 10% serum media and plate. Fibroblasts will be the only cells that stick and grow.
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