0
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.
