Help with endothelial cells - Endothelial Cells Culture (Oct/19/2005 )

Hello
I'm having some problems culturing porcine endothelial cells. I isolated them and they look healthy, very good morphology, after I passaged two or three times, they start to lost their cobblestone shape and become elongated.
I tripsinize them (Tryp 0.05) and keep them in M199 5% FBS
Tips Anyone????
Yalpizar

They are normal fibroblast?

Sometimes they do that....

hi,
primary endothelial cells are very sensitive cells.
please tell more about the medium you are using: glucose, growth factors, etc... or maybe this is a ready medium?

Hello
I've using M199 (Invitrogen) and FBS Characterized(Hyclone). Now I'm trying a diferent medium, is called Endothelial Cell Growth medium, and of course, I don't know the recipe, only that is fully supplemented with Serum and Antibiotics.
I want them to keep their cobblestone morphology, but they always change to elongated cells.

the fully suplemented media (the last one you use) is probably the best for primary endothelial cells.
i suggest that you will check additional endothelial properties and not only morphology. for example: ldl uptake, vW factors, TEER, and others.
it is known that primary endothelium tends to loose its properties during in vitro culturing. if you lucky you will keep some of their properties...