I am currently in an internship in a virology laboratory : I make my cells cultures in a "clean" room, without viruses and a fex possibilities of contamination. I thawed my cells and I trypsinized them 6 times (6 passage until now so). During the last month, they have been fine : into DMEM, with Foetal bovine serum, antibiotics (gentamicin and penicillin), HEPES buffer, L-Glutamine and of course MEM. I have let them during a week while they were sub confluent. When I came back on Monday, a huge part of was in the culture medium. I tested the culture medium for mycoplsam : it was negative (no bacteria).
The strangest this about that is that I sowed wells with the exact same cells suspension, and the cells were normal (no cell in the culture medium). The problem might come from the flask I guess (the flague cap must stay open to allow CO2 passage)... I would want to get advices about what happened and how can I avoid it in the future.
Thanks for the time allowed and your consideration
Johann PONS
After a few days, I managed to save my BHK21 cells : in a medium flask, I poured the surnatant in a 15 ml tube. I centrifugated it at 1000 rpm, 5 minutes, 20C. I gently removed the supernatant from the tube with a micropipette and resuspended the bottom with 7 ml of new BHK21 culture medium and transfered it into a small flask. I finally added 1 ml of Foetal Bovine Serum to increase their growth rate. 3 days later, the cells were confluent, they were stuck to the bottom of the new flask.
I saw that I still hasd a few cell in my
medium flask that were attached to it : I trypsinized them as I would have do for normal BHK21 and I put them in a new small flask with 7ml of culture medium and 1 ml of Foetal Bovine Serum. Their shape is sightly better than the surnatant cells after 3 days at 37C.
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