I am going to transfect AAV293 cell with the AAV plasmid by adopting the PEI protocol.
I wanna ask what is the best way to collect the viral stock ? In online literature and methods there are numbers of confusing methods.
Some mention it better to collect the supernatant after 48 hours, some mention its better to collect supernatant after 6 hours of transfection when you change the medium and also 48 hours after transfection. However, some mention that after 48hours it better to scrape the cells into growth medium with cell lifter and than collect the supernant ?
Last time i do transfection and collect the supernatant after 48 hours, but i didn't get anything. however, that was my first time to transfect the cell. so maybe contamination or lack of experience was the factor.
But this time i am not ready for any mistake and need to get high titer of virus. Yours suggestions will be highly appreciated.
Dear Waqas, why would you colledct supernatant after transfection, most of AAV virus is reserved in cells. The common protocol is collect the transfected cells after 72h, freeze and thaw 3 times. Ultracentrifugation is recommended to purify AAV.
Genemedi is expert in AAV production and genetherapy, you could find more information on this website: www.genemedi.net/i/aav-packaging
Dear Florian thank you so much for your nice feedback.
I wanna ask, i will change the medium 16 hours after the transfection. So should i also need to collect that supernatant ( which i change after 16hours) or i should just collect the supernatant after 48hrs?
Thanks.
Dear,
All will depend if the collection and concentration method used, iodixanol or CsCl. After transfection of the cells (18 x 150 mm dishes with a confluence of 70-90%), you don't need to change medium before collecting. I personally collect medium and cells 72 h post transfection. For purification, freeze and thaw x3, benzonase and short centrifugation for getting a solution without cell debris. Continue with iodixanol protocol and finally concentration using 100k filter device. Titer around 1x10e13 vg/ml in 400 ul.
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