Help! Large vacuoles observed in HEPG2 cell culture! - (Apr/05/2007 )

Hi, I'm in the midst of culturing HEPG2 cells right now, and I am starting to observe the formation of many vacuoles in the cells, some are huge, and some cells have many tiny vacuoles visible under low magnification in the microscope. Anyone knows the possible cause of this or have any recommendations? This definitely doesn't look normal. I'm currently culturing the cells in DMEM with high glucose.

Thanks!

I think I used to add insulin to the media-my PI told me that it reduces the formation of the vacuoles. Unfortunately I don't know the concentration I used.

We dont see these structures. Rather cells form 3-D clusters at high density. 10% FBS DMEM/high glucose, no insulin added.