I have cultured NIH 3T3 fibroblast cells on soft ligand-coated polyacrylamide gels. The polyacrylamide gels were (12% acrylamide 0.6% bis-acrylamide) coated with fibronectin (100 ug/ml). The cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin at 37 C, 5% CO2 and 90% humidity. After 10 days of incubation with several change of medium, the cells did attached to the gels surfaces however without differentiation, keeping their circular shape. Do anyone know what could cause that absence of differentiation?
Thank you
Hello, Diane.
Did you try plated your cells on glass, only? Without coating!
You can to compare both shapes. Probably, cells do not adhere on coating that you make for reasons as incorrect composition or time.
Good job!
Hello Marvin,
Thank you for your suggestion. I have plated my cells on petri dishes as well and they proliferate and differentiating correctly. However for this study, I want to see those cell behavior on softer surfaces such as hydrogel. They seem to adhere to coated hydrogel surface however without differentiating.
Have you checked the cells' gene expression? It is possible you are seeing a response to your ligand that is producing the cell morphology you describe. We tend to see the round shape coincide with increases in expression of alpha actin. Interestingly, there's an older paper that discusses the role of alpha and it's cycling. Since you are attempting to grow the cells on fibronectin coated polyacrylamide, the cells may be having difficulty attaching due to loss of alpha 5 expression, which is a receptor for fibronectin. Obviously if you are using some other ligand that would potentially change what you are looking for, but I'd start with differentiation markers.
Hi Diane.
Your cells may be transformed after ten days of culture, explaining why they look like undifferentiated cells. If you are looking for normal cell behavior you should adjust your experiment to shorter periods. How much cell adhesion occurs after 1 hour, 12 hours and 36 hours? Have you tried to increase fibronectin gel concentration? What about using vitronectin or laminin as ligands? I would try this before think about gene expression.
Thank you to both of you for your suggestions,
they are really helpful.
I will seed the cells on PA-fibronectin gels for 12h and 36h and then see if they have similar behavior than on plate. I already increased the fibronectin concentration, however that could be a good idea to try an other ligands as suggested by Newton. If none of those solution works then I will tried Nathan idea and look in to the cells gene expression.
Thanks again,
I will keep you posted,
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