I am cultivating RPTEC isolated from mouse renal cortex and immortalized with temperature sensitive SV40 (tsA58) in DMEM:F12 with 2% certified FBS (Gibco 16000-044), 2 mM L-Gln, 1x final insulin transferrin selenite (Sigma I-3164), 10 ng/ml EGF (preprotech AF-100-15), 4 ng/ml T3 (Sigma T-6397), 10 nM Dexamethasone (Sigma D-4902) and pen/strep.
Dishes and flasks are TC-treated from corning and coated with 0.1 mg/ml Rat Collagen I in 0.12% AcCOOH (8 µg/cm²)
Medium bottles are kept at 4°C and don't last for more than 10 days.
After a few passages some lines start to have a fibroblastic appearance while other keep their epithelial morphology.
Does anybody have suggestions to solve this?
Thank you for your help!
And I'd add how to understand that the same cells seeded in a 150 mm dish for exp don't look right whereas they do in a 75 cm² flask for passage (lines 1671 and 1673 on page 4).
Hi Daniel Pouly . The problem could be with the collagen coating. Try denatured collagen (gelatin) or serum coated plates.
Hi Steingrimur Stefansson ,
Thank you for your answer. I'll try to coat differently and try to figure out how a difference in coating efficiency could vary on the same (at least as catalog says) TC-treated surfaces of corning dishes and flasks.
Best,
Daniel