Hello Lucian Miles

While the presence of vacuoles in Vero cells is associated with aging, it is not a definitive indicator of aging itself. You should look out for other signs of aging which includes increase in cell size (being larger and flatter), change in cell shape, nuclear enlargement, increased organelle size and number (like lysosomes, mitochondria, and the Golgi apparatus can become more prominent), besides accumulation of vacuoles in the cytoplasm which is a common feature.

Vero cells may be undergoing senescence which is the process of biological aging. These cells are known for their susceptibility to vacuolization. Presence of vacuoles in Vero cells may indicate either infection or cellular stress. You should run the beta-galactosidase senescence assay, specifically the senescence-associated beta-galactosidase (SA-β-Gal) assay, which is a widely used method to test whether Vero cells are undergoing senescence.

Best,

Lucian Miles, I would say, only take it as a cue that your cells are aging but keep in mind that it could also be a number of other factors. Other signs or aging include reduced proliferation, which you could test by BrdU staining and flow cytometry. However, trying to test if your cells are aging could be significantly expensive and time consuming. My recommendation would be to thaw a new vial from a low-passage stock, begin tracking passage numbers consistently, and note when you start observing signs of aging. That passage number can then serve as your empirically determined limit moving forward.

All the best!

The presence of vacuoles in cell culture can sometimes suggest that the cells are aging, but it is not always the case. Aging cells, also called senescent cells, often become larger, flatter, and may develop more vacuoles. These cells also divide less and may show specific aging markers like β-galactosidase. However, vacuoles can also appear due to other reasons such as stress or damage. When cells are under stress, they may start a process called autophagy to clean up damaged parts, which leads to the formation of vacuoles. This can help the cell survive, but too much of it can be harmful. Sometimes, vacuoles form because of increased lysosomal activity, which is common in aging cells but can also happen due to exposure to drugs, toxins, or infections. Infections like mycoplasma or viruses can also cause vacuoles to appear. Large or irregular vacuoles may also be an early sign that the cell is dying. To find out if vacuoles are related to aging, it's important to look at the shape and behavior of the cells, test for aging markers, check how fast the cells are growing, and make sure there is no contamination. So, while vacuoles can be a sign of aging, they are not a sure sign on their own.