I am working on LA7 cell line which is rat breast cancer stem cells. my culture media contain 90% DMEM and 10% FBS and 1% P/S. these cells are in passage 16 and at confluency about 50% , they were growing normally and as usual they rarely contained small vacuole-like masses; but at confluency about 80% these masses grow up and now these cells have big and somewhat giant vacuole-like masses as i mention them in the picture, i wonder what are they exactly?
Hello Ashkan Garshasbi
The presence of several large vacuoles in cells is a sign of stress. You may consider the following.
1. At 50% confluency, they grow normally, but at 80% confluency, these vacuoles grow up and become giant vacuoles which means that when cells are too dense, it leads to intercellular competition for nutrients. The lack of nutrients in cells leads to starvation-induced autophagy and vacuolation. To ensure sufficient nutrients for the cells, you may check your media composition, and if needed, you may add extra supplements to the growth media like 2mM L-Glutamine and 1mM sodium pyruvate.
2. Check for changes in osmotic pressure and pH value in the media. Replace with fresh culture media to adjust to the appropriate pH value, and then culture.
There are other factors such as,
1. Long-term trypsinization time may damage the cell leading to vacuolation. Also, long-term existence of many air bubbles in culture medium which is caused by excessive force during pipetting can also lead to vacuolation of cells. Choose the appropriate concentration and digestion time for trypsinization and prevent too many air-bubbles by avoiding blowing too hard during the culturing process.
2. Check for mycoplasma contamination. Vacuolation often occurs due to mycoplasma contamination.
Best.
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