Dear Neha,

What you have observed is consistent with what I have heard from many of my colleagues who engage with such in vitro monolayer studies. Cells are highly sensitive biosensors & adapt to slightest of the changes (literally any change!) in their vicinity or growth climate. Therefore, I am not surprised to learn your observations. The problem is also aggravated by the fact that a shockingly high number of in vitro cell lines (>90%!) including Caco-2 are contaminated now. Therefore, standardization with these type of co-cultures or even mono-cultures is very difficult & hence, in my group, I have stopped working with such systems.

Regards, Sourav

Dear Sourav, 

Thank you very much for the answer. But I have been working with this model for sometimes, and my colleagues have been using the same model for many years, and they have never had such problems. I have used the exact same protocol and reagents they have used, but I have now started to observe such changes. So, I am not sure what might be the reason. I, however, do agree with the high density of cells that might be a cause. But I am unable to understand what caused such changes. 

Thanks again! :)

Dear Neha,

You are right. However, that factor of predictability on when such morphological changes will begin to appear is quite difficult to have. Even minor things trigger changes. Let's hope you can resolve these issues with developing fresh cultures from new cell lines.

Regards, Sourav