Hello everyone,

I am working on establishing primary cultures from human lung cancer tissue samples (patient-derived cells, PDCs). A common challenge I face is the mixed outgrowth of both epithelial (presumably tumor) cells and fibroblasts in the same flask.

I have tried differential trypsinization based on the literature (using 0.25% Trypsin-EDTA for short exposures expecting fibroblasts to detach first). However, in my hands, the fibroblasts appear to be more trypsin-resistant than the epithelial clusters, contrary to many protocols. This makes the method ineffective for my specific cultures.

I would greatly appreciate advice from the community on alternative or optimized techniques. Thank you in advance for sharing your expertise and experiences. Both literature references and practical "lab hacks" would be incredibly valuable.