I make lentivirus weekly, but just use 30% of total volume. the leftover is aliquoted as 1 ml each tube then freeze at -80 freezer.
Questions:
1. is this store method correct? directly put in -80 degree.
2. How long can the fresh lentivirus store at 4 degree fridge ? (Do not need too much frozen batches and wonder learn how to keep fresh virus.)
I would be grateful if you could give me any help and advice. i would appreciate if you could explain reasons or recommend relevant paper or protocol !!!
You can freeze lentiviral vectors, as straight supernatant from producer cells or after any method of concentration. Freeze rapidly and store at -80C.
Freeze-thaw cycles definitely can affect your titre, especially of concentrated vector where we have seen the titre drop by up to 50% (so we always titrate vector after freezing first). It is important to consider your envelope too; VSV-G is relatively stable and so you can concentrate by ultracentrifugation and you can freeze it. Others are not so stable and will fall apart on spinning and a freeze thaw will have worse effects.
freeze at -80 in small aliquots and use each tube just once. For short term storage, keep the vector at 4degrees (fridge) for 3 or 4 days, rather than freezing and thawing, but for longer storage definitely store at -80.
I've always been taught that, for every 24 hours viral supernatants are kept at 4degC, you should expect a ~15% drop in titre. So it's best to aliquot and freeze at -80degC as soon as you can.
thanks for the answers! That makes sense! So summary: after haverst virus, freeze lefover rapidly(as soon as possible) at -80 degree.
Dors “The rapidly freezing ” mean i must use liquid nitrogen? Or just freezer?
Thanks !
I just put them straight into the freezer and it's fine. I don't flash-freeze, although I've never compared the two nor have I seen any data about that.
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