Hi Ilana,

With the methods of preparation you are describing. You have lysed the cells, filtered off the clumps and add the lysate directly to the cells. The lysate contains a high contents of virus and other components such as glutamate, protease and various intracellular proteins and signal molecules. Depending on the concentration, they can be very toxic to the cells. Additional purification steps (usually ultracontrifugation) will be necessary to eliminate these factors. AAV with G-CaMP6 is not your problem here.

I second that. You need to purify the virus, which is, on a small scale, most easily done by iodixanol gradient purification and dialysis.

There could be few possibilities.

1.GCamP6 might not be contamination free.

2.Filtration assemble might not be well sterilized.

3.If you look for the type of contaminants, this may reveal the source of contamination.

I tried the same by infected the primary cortical neurons on day 1 with crude extract of AAV EGFP virus. Even after 5 days I did not see any expression of EGFP and the cells looked not happy as mentioned by others here. You must purify the virus to test it, in a way later on this will save a lot of time. Iodixanol or CsCl2 ultracentrifugations can be done. or you have to use some kits like

http://www.cellbiolabs.com/aav-purification

Hi Ilana,

if you want to avoid purifcation, you might want to try using the AAVs found in the cell culture medium (instead of using cell lysate). AAV9 gets secreted quite efficiently and should work well for the transduction of primary neurons. Just transfect your 293 cells as usual, exchange medium 6 hours after transfection. 72 h after transfection, harvest the medium and centrifuge it at 20000 g. Take the sup and add 1/10 volume of PBS-MK + 1 M NaCl. If you like you can determine the titer via qPCR or just use this mix for transduction of your cells straight away. Caveat: I have not tried this myself yet, but some people in the field have successfully used this approach... Good luck!

I agree with the above, ultracentrifugation is likely the answer.

When infecting primary neuronal cultures with unpurified crude AAV preps it is important to not add to much volume of cellular virus containing extract to your cells as this cell lysate is toxic at a high concentration (You can use AAV free 293 lysate as a control for toxicity). For your crude AAV infections I would add at most 10% of your culture volume which will limit the MOI you are able to use. We have been able to keep primary mouse trigeminal neuron cultures alive for >2 weeks after AAV infection when we use less than 10% of the volume of AAV. If you need to go higher with the MOI you will need to purify and concentrate your AAV as described by the others above (I prefer iodixanol since it is quicker than CsCl). For neuronal transduction it may take a while to see any transduction in vitro if you are using ssAAV vectors as described by Jonathan above. Your AAV serotype will also influence transduction levels greatly as some serotypes work poorly in culture. We like AAV1, 6, 7 or 8 for primary neuronal infections in vitro, although these may not be the best serotypes for hippocampal neurons. To screen which serotype works best you can do quick pre-screen with a serotyped scAAV vector.

You need to purify your AAV. For how to purify, please refer to:

1. Hauswirth WW, Lewin AS, Zolotukhin S, Muzyczka N. Production and purification of recombinant adeno-associated virus. Methods Enzymol. 2000; 316:743-61.

2. Grieger JC, Choi VW, Samulski RJ: Production and characterization of adeno-associated viral vectors. Nat Protoc. 2006; 1(3):1412-28.

As I am trying to avoid virus purification as well I like to follow the steps described above by Benjamin Strobel.  I am just wondering if you are using a ultracentrifuge  for the 20,000g centrifugation and for how long you centrifuge? I got confused because i actually thought that via ultracentrifugation you pellet the virus instead using the supernatant?

I use AAV9 in vitro for transducing DRG neurons and it works well. I have settled on a kind of hybrid purification that doesn't use ultracentrifugation but it cleans up the virus better than just crude lysate. As Benjamin Strobel mentioned, AAV9 is secreted well into the medium. So I take all of the medium and the cell pellet (3 freeze thaw cycles) from transfected HEK cells (5 days in serum free medium) and use this. I concentrate and semi-purify by using an Amicon 15K 100KD MW filtration column. This can reduce my ~15 ml of medium/lysate to ~150-250 ul. In the process, I do a PBS buffer exchange. In this way, most proteins (under 100 KD) and metabolites, small molecules, etc. are removed. There are still some larger proteins, but overall the cells look good. I don't get any cell death. Give this a try. 

We have developed some AAV purification kits that might be useful to you. Currently, we have 2 different scales for AAV purification: mini and midi. Our mini kit can purify from between 0.5 mL to 10 mL of cell culture media, cells, or the two mixed together. The midi kit can purify from 10 mL to 50 mL of similar input. I have found that as little as 0.5 mL of mixed cells/cell culture media input can lead to visible expression of an alkaline phosphatase transgene in vitro.  Only standard laboratory equipment ie, a swinging bucket centrifuge, microfuge is required, no ultracentrifugation necessary. The process is rapid (1 to 1.5 hours) and multiple purifications can be done in parallel to accommodate large screening experiments. We are also working on larger scale purification (50 mL to 1 liter or more).

Please send a message or visit our website for more information:

https://norgenbiotek.com/category/virus-purification

Best of luck!