Don't know why my cells are unhealthy - (Jan/26/2012 )

Hello everyone. I am in desperate need of help. I am growing CHO cells stably transfected with the human hERG ion channel. I've been growing these cells successfully for the past 3-4 years with no issues. All of a sudden, the cells crashed! After passing them, only a few cells attached and barely divided. I have tried several growing conditions, tested CO2 & temp, tested for mycoplasma and finally narrowed it down to be what I believe is an issue with our medium.

The vendor we sourced these cells from recommend growing them in Ham's F-12 + 10% FBS with double selection: 750 ug/ml zeocin and 10 ug/ml blasticidin. I do not add P/S. Our frozen stock has been in use for 3 years so I am sure they are OK. The cells come out of thaw looking healthy, but after passing them, they look terrible! Only a few adhere to the flask and if they divide at all, it is very very slow growing. To get them to grow at a normal rate, we have lowered the zeocin concentration to 400 ug/ml, but the cells still look unhealthy (several senescent/multinucleated cells) and assays produce only mediocre results.

If I remove selection, the cells grow normally. It seems that there may be some sort of negative interaction with the base medium and selection that make the cells unhappy. My colleague recently brought up CHO-M1, which she has growing in Ham's F-12 + 10% FBS (different brand & lot from the CHO-hERG above) and 100 ug/ml geneticin. They also have the same abnormal cell morphology the CHO-hERG cells have. They only thing in common between these two cell lines is the Ham's F-12 they are grown in. They are in different incubators. Between my colleague and I, we have about 10 different cell lines in culture and only the two in Ham's F-12 have this issue. We have also tried a different brand of Ham's F-12, but we did not see any improvement. For all I know, the two vendors could be sourcing their raw materials from the same place.

Any thoughts on what my problem could be? Anyone ever encounter an issue like this before? I've called the medium vendor and cell line vendor and neither have been able to help me.

You could try out a completely different medium formulation. CHO grow well in RPMI1640+10% FBS for instance. Have you changed your batch of FBS?
You could try and cycle your cells (say 1 passage in Zeo followed by 1 in Bleo and see if the cells grow better. I have experienced that some cells do not "like" double selection and you may need to a checker-board titration of both antibiotics.

Some cells frozen take a while before they grow at the normal rate. Don't start with the selection agents for the first 1-2 weeks, then add them in, when they are growing fine.
These are stable cells so they will not deselect without selection agents for a few days.