I was keeping HEK 293T cells for an AAV production, although my last mycoplasma test was positive. I already discard the cells in order to avoid more contamination in our culture room, but I would like to hear some opinions about the real ¨danger¨ of mycoplasma in cell cultures that would be used for AAVs production (research purposes: i.e animal experimentation).
Thanks!
I don´t know what test you use for mycoplasma. Sometimes, PCR method is false positive. You should test Hoecht Method. Mycoplasma could affect cells grow and biochemistry experiments.
Cheers
Because you have mycoplasma in your cells, you can never completely trust your results, as the observed results could also occur due to the mycoplasma contamination. In case you are studying some pharmacological inhibition or immune response in mice, mycoplasma may supress or enhance a particular phenotype and you will never be completely sure of what is happening.
get rid of mycoplasma!!! You will probably increase your virus yield, and sleep good at night considering that your result will be more reproducible!!!
cell culture should be mycoplasma free, it does affect the yield of virus propagation and your experimental results can be reproducible
How did you purify your AAV ? Ultracentrifugation, Iodixanol or with kit ?
Best way to test the possibility of your AAV stock with mycoplasma contamination would be to infect fresh HEK293T with your doubtful AAV and culture for 3 to four days and test the mycoplasma contamination. But include also one extra HEK293T well as a control. May be this would help you in faster way.
If you find out that your AAV got contaminated with mycoplasmas, then best way is to prepare a new stock. Where do you want to inject the virus in animals ?
Good luck.
A lot.
Beware of mycoplasmas and check your culturtes regularly (each month ??)
Working with anything contaminated with mycoplasma is always a complete waste of time as their impact is so unpredictable. In your case the unpredictability would be the exact nature of the AAV stock produced or the effect of any contaminating mycoplasma still present. Either factor would make results too difficult to interpret or reproduce with confidence. The other danger is to reputation. Those seen to be working with mycoplasma contaminated material run the risk of permanent scepticism. Good call to ditch the cells.
Thank you for your replies. I´ve been very busy these days and finally I have the time to answer you back in a proper mode. At the present the myco issue is solved and cells are safe and producing! :). Sergio, the kit that I use is the Mycoalert Mycoplasma Detection Kit (Lonza). Goutham, I purify the AAVs by ultracentrifugation (iodioxanol gradient). I already performed an infection test with my last AAVs and it´s ok, but I will perform a new infection test with some others AAVs to be sure of all my AAVs stocks are clean. The injection will be performed monkeys brain. Thanks again to everybody!
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