Dear All,

I am currently trying to establish cell line for canine cutaneous epithelial tumors (hair follicles in origin). For primary cultures, I followed the standard protocol using 10% FBS in DMEM containing 1%Penicillin-Streptomycin. I subcultured it using the same media as above. At P3, I performed immunocytochemistry and found that most of my tumor cells were strongly positive for vimentin and nestin but showed very pale positivity for pan-cytokeratin. The secondary cell line did not survived until the fifth passage (I tried twice but the cell stop growing and started dying at around 5th passage). The DMEM contained: 4.5 g/L D-glucose, L-Glutamine and 110 mg/L Sodium Pyruvate. I had look into some journals reporting the establishment of squamous cell carcinoma cell line which used 3T3 Feeder Layer, cholera toxin and hydrocortisone. I would like to ask if anyone can share their experience in culturing tumor cell lines of epithelial in origin and the basic growth supplements and suitable media required for successful cultivation of epithelial tumors. Thanks a lot !