We have used PEI Max 40KDa to transfect 293T cells for the production of AAV for several years. But in our experience, whether AAV released into supernatant is independent of the transfection reagent but the serotypes, about a third of AAV8 and AAV9 are secreted during the production in 293T.

We are experienced in AAV production. You could find more information and AAV manual on this website: www.genemedi.net/i/aav-packaging

We use PEI routinely for tripple transfection of HEK293 cells. It works very well and is far less expensive. I would recommend. Sophia

Hi Henning, Have you been able to adapt the baculo virus system for use in your work for making AAV? Is this practical for a normal academic lab? If you can share any information about how to set this up (protocol or reference, etc) that would be great. I know that companies like Virovek use this system and generate super high titer AAV...but it is presumed this is large scale and not easily replicated in a typical lab setting.

I should also add that I routinely use PEI for transfection of many cell types and it works very well and is far cheaper as Sophia pointed out...which is why I am trying to adapt this for the AAV production instead of lipofectamine 2000 or equivalent, I would like to focus on use of PEI transfection in AAV packaging and how much virus can be collected in the medium vs cells for various serotypes. Perhaps other people have experience with this and may know some tricks to get higher virus yields in small scale runs (low cost for routine testing). Thanks.

We have used PEI/plasmids in HEK293 cells and baculovirus in Sf9 cells to produce AAV serotypes. You may refer to the following:

Journal of virological methods 139 (1), 61-70

Journal of virological methods 144 (1), 32-40

The journal of gene medicine 12 (2), 157-167

Biotechnology progress 21 (1), 154-160

Although we have not optimized the production parameters for the PEI/plasmid technology, the specific production of AAV were comparable to baculovirus system.

Late harvest is related to the release of AAV due to low viability but still majority of AAV remain associated with cell debris or entrapped in the viable cells.

All the best.

Henning,

Thank you, this is all very helpful. I am going to look into some info on the Sf9 cells and media, etc for getting set up.

Does it make a difference using AAV293 cells versus standard HEK293 cells for AAV packaging? The AAV293 are supposed to have more surface area and are said to yield higher titers of virus assuming the cells are not grown beyond 50% confluence during expansion. Does anyone know what is unique about the AAV293 cells?

I am trying this PEI triple transfection right now and immediately I noticed that the AAV293 cells are starting to detach to some notable extent after only 24-36 hours. I assume this is because the PEI is a bit harsher on the cells than other transfection reagents I have been using (L2K)--which may be consistent with viral particle release into the media at later times being due to cellular stress. I am using 50uL of 1mg/mL stock of filtered PEI Max 40KDa from Polysciences per 10cm plate, or the same amount of PEI linear 25KDa from Polysciences per 10cm plate. I previously tested and found that 40-60uL PEI gave excellent transfection of a 10cm plate of nearly confluent AAV293 cells, so I went with 50uL to be in the range. Any ideas how to deal with this? Is gelatin water pre-coating a good idea?

Does PEI triple transfection work well for harvesting AAV from the cells at 72 hrs post transfection or does this method cause too much release and cell stress even at this earlier point in time?

Hi Jonathan, I am also trying PEI triple transfection protocol and I have the same problem, i.e HEK cells start to detach after 24-36 hours. Do you (or anyone) have a solution for this?

Hi guys, we use PEI to make AAV2/2, 2/5 and 2/8. We find quite a difference between the serotypes. 2/2 is far more lytic and the other two and prob needs to be harvested after 36 h. However, we tend to leave 2/5 and in particular 2/8 for 72 h as the yield is higher and our HEK293s cells tolerate this time frame very well.

Hey guys, I have been working with PEI in different MW in the past. PEI is either Linear or Branched, it can be made in different Molecular Weights, from 1kDa to higher to 100 and more, by increasing the MW, Cytotoxicity is increased, Branched one is also more toxic than Linear, if you see detachment of cells, that is normal, because PEI is toxic, more than 24 hours incubation with PEI complexes, is not good for cells. The best one ever worked in my hand, was Linear PEI 25kDa and incubate for 24 hours or less, uptake in within few hours, even 4 hours incubation can work

Sorry, I should have clarified. While we leave the HEK293 cells for up to 72 h post transfection prior to harvest, we do replace the medium after 5-6 h with fresh serum-free medium. PEI is indeed a little toxic; best to remove.

how long do you keep the cells in serum free mefium? i guess not more than few hours...since cells need serum...beside the discussion about PEI, I am also interested to knoe best cell line for AAV production: normal HEK293, AAV293, HEK293sG or HEK29T cells which are used for Lentiviral vector production. thank you for your comments.

my writing mistake, sorry..HEK293T cells

No, actually we keep the cells for 70+ hours in the serum free medium. I think that this is the trick. My theory is that when the cells don't have serum, they stop trying to divide. I know that since they are very confluent at the time of transfection, growth is already somewhat arrested. However, growth is even more arrested when the serum is removed. This gives the cells more time to make AAV before becoming over-confluent and as a result dying. So keeping the cells less happy, buys them more time for AAV production. Neat I think!

I have had similar experience to what Sophia and Amir have discussed above. I find that linear PEI 25KDa from Polysciences works well, although I get slightly better results with PEI Max 40KDa from Polysciences. I too change the media for low (1%) or no serum AAV293 media at 1 day post transfection with PEI. I then allow virus production to proceed out to a minimum of 72 hrs or ideally out to 120 hrs to allow for a late harvest with a good portion of the AAV particles being released in the media. I found that even when the cells detach and look bad at harvest time I could still get high titer of virus. I actually prefer the late harvest with the cells starting to detach because it makes it fast and easy to collect up the cells + media without trypsinization or cell scrapers.

Hi, I'd agree with others as to the fact that PEI-DNA is toxic to cells. Usually media is replaced after ca.6hours and cells collected at 72h. 293 grow in a standard high glucose DMEM/10%FBS and I can tell this is good for AAV5. Some people in the lab keep cells in 1% serum in agreement with other comments posted. Lab where I make my vector works with all serotypes, my guess they follow the same procedure. Hope this helps.

I am really curious about the timing of harvest. Is longer time outputs higher titer or is it depends on the sterotype of the AAV you want to make? In my case, I change serum-free medium contains PEI with the full medium after 5 hours transfection and harvest the virus ~60 hours later. Does the shorter time explain why my AAV titer a little bit lower? around 10^12?

I have some trouble in concentration of the virus infection cells growth media. Anybody has good idea? Will NH4SO4 work?

Anybody can give me a detailed protocol for AAV triple transfection?

Hello, I'm transducing trigeminal neurons with viral particles AAV5. There a way to better the efficiency of infection?

I tried Transporter 5™ Transfection Reagent (http://www.polysciences.com/default/transporter-5-transfection-reagent) to make AAV (AAV-DJ). After 3 days culture, harvested cells, purified AAV by purification kit. Then run PCR to check target gene. It seems no virus generated! At the same time, I also did transfection by lipofectamine LTX with PLUS reagent, it worked very well. Why?

I have used PEI transfection for rAAV production. I observed two important things

1. How you prepare the PEI? Different people have their own ways. PEI is not easy to dissolve and some recommend adding acid to escape this hurdle but I observed that my cells were completely unhappy when I used acid. So I left my PEI @80c water bath for 30-40min and this makes life easy. Cells were happy and result was good.

2. The ratio of DNA to PEI. You can find different ratios from 1:1 to 4:1. Try different ratios and check that works best with your hands. For me DNA to PEI ratio of 1:3 was great.

Good luck

AAV293 cells are derived from the 293 cell line, and they can be transfected with reagents for the 293 cell line (e.g https://altogen.com/product/293-transfection-reagent-emb-kidney-crl-1573/). With regards to virus production and harvesting, as long as you have the cells in a good medium and don't be too harsh on them during transfection, you should be able to get good results.

The PEI I used is a solution stock directly came from the company. And the ratio of total plasmid amount (3 plasmid together) to PEI (1 mg/ml) is 50 ug to 150 ul.

Similar plasmid amount was used by LTX transfection, it worked very well.

We have used PEI Max 40KDa to transfect 293T cells for the production of AAV for several years. But in our experience, whether AAV released into supernatant is independent of the transfection reagent but the serotypes, about a third of AAV8 and AAV9 are secreted during the production in 293T.

We are experienced in AAV production. You could find more information and AAV manual on this website: www.genemedi.net/i/aav-packaging

Here's an AAV protocol where you can find lots of details about AAV transfection. Hope it can help you Jonathan.