Hi Sultan, 

I would avoid medium to turn yellowish as at such pH virions are high unstable. I would add HEPES or reduce production time prior to harvesting. Also try reducing confluence or use milder trasfection reagents (I use effectene or Calcim phosphate). As an alternative, you could harvest at 24 hr, change medium and do a second harvest at 48hr, then reunite supernatants. 

 I also produced virus in optimem without much observing any difference. 

If not, you could use 293T HEK strain that express antigen T, thereby greatly increasing virus production. 

I routinely produce 10^6-10^9 TU/ml by simple concentration.

Hope it helps

Francesco

As Francesco suggested, use HEPES in your media to keep the pH stable and your virions happy. Lipofectamine is more toxic than PEI (for example) but when I compare the two I didn't really see much difference in titre. I harvest every 24 hours as the virus has a half-life of 8-9 hours at 37 degrees.

However in my experience, the MOST important determinant of virus titre is the transfer vector itself. Some vectors always produce well, and some (particularly some of the older ones) produce horribly. If you're using a poor producer you may have to scale up your production.

Also, keep aside some supernatant before your UCF and use that to infect cells as well - this will tell you how much virus is lost when you centrifuge. I usually use 17 000rpm but I'm not sure if that would make a difference in your case.

Low pH is not suitable for viral particles, so try to start with lower confluence (60-70 %) Replace trasfection media after 8-12 h. Harvest the media 48 h and 72 h post-transfection, pool and centrifuge.

24 h incubation in 4 degrees may cause reduction in viral titer. I suggest you to check if 1-2 h incubation can increase the titer.

I'm agree with Francesco that you need to use 293T cells but I think milder trasfection reagents such as calcium phosphate may reduce the transfection rate.

OK I just re-read your description and I have questions: How are you titering your virus? And what system are you using to express GFP and your GOI? IRES, 2A, separate promoters etc. When you leave your tubes upside down for 3 minutes, is there any liquid left on the pellet? You don't want to pellet to dry out or the virus will be inactivated.

Thanks for the suggestions, everyone! I'll try adding HEPES and maybe pyruvate to the buffer. I actually tried earlier to transfect at ~70% confluence and did a wash-off with fresh media replacement --> in this case, the media didn't turn quite so yellow (maybe orange-ish), but the titer was even worse then! That's why i transfected at higher %confluence.

Also, the cells are indeed 293T (sorry I did not make that clear in my answer).

Jill - the transfer plasmid is a modified Cas9 driven by EF-1alpha promoter. It is bicistronic with EGFP (separated by T2A element). After transfection, I checked the cells every time I've done this, and they look very bright green.

There is no liquid left on the pellet when turning over. I followed the lenti production video on JoVE for that part: http://www.jove.com/video/1499/lentivirus-production

For this last run, I just visualized transduced HEK cells with fluorescence microscopy. I'm about to re-titer the same vial with FACS to confirm it. I did FACS in the past and got horrible yields (~2% transduction, very low titer).

Also, one other thing - I actually did keep a supernatant fraction (right after spinning down debris but before syringe filtration and UC). Oddly enough, the titer was worse than it was with concentrated virus (each normalized to total volume, that is)! Now I'm thinking it could be the serum, left-over transfection reagent or the low pH that kills them while they're in pure media... does that sound possible?

Ah. Bacterial proteins such as Cas9 and Channelrhodopsin often result in much reduced LV titres, about 10-fold in my hands. The transfection will indeed look very good but I usually get titres of about 1e7 to 5e7 IU/ml from one T-75 flask, which is about 10-fold less than I would normally expect.

Try scaling up your production from one T-75 to (for example) three T-225 flasks - that's a nine-fold increase so you should then get something approaching a 'normal' titre.

I have a lentivirus protocol from my labmate who is a virology expert. To make 293T cells adhere better and stay happy for 3-4 days, we often coat the plates with 0.5 ug/ml Poly-D-lysine for 1 hr at 37C, then wash once with water. We plate ~5.5 million 293T cells in each 10 cm plate and let them grow overnight before transfection. You can mix lipofectamin (I use lipofectamin 2000) with the plasmids in OptiMEM, incubate for 20-30 minutes and add them into the cells in fresh growth medium. It is not necessary to use antibiotic-free medium. I often change the medium after 5 hours and change again the next evening (~36 hours). 48 hours after transfection is the best time to harvest the virus. I add new medium and collect the supernatant again 6-8 hours after that.

We spin the supernatant at 2,000 rpm for 7 min, at 10ºC to remove cells/cell debris. then we pass the sup through 0.45um filters into Amicon15 ultra (Cat # 24 per pack: UFC910024; 96 per pack: UFC910096), for each set, 35 ml (from 5 plates) into two Amicon tubes. We centrifuge the tubes at 2,500 rpm for 25min. We use these Amicon tubes to concentrate the virus. It is much easier and faster than ultracentrifugation.

You can then use fresh virus to transfect the cells (we use 0.5 ml of concentrated virus for 0.5 to 2 million cells in suspension, depending on the cell type) or freeze concentrated virus on dry ice and store in -80C. For most cell types, you should add 8 ug/ml polybrene together with the virus. Non-adherent cells such as lymphocytes and leukemia cells require centrifugation for at 2000 rpm for 2 hrs at 37C. We incubate the cells with virus overnight and change the medium.  

Dear Faraz,

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Regards,

Nicolas

Microscopy is not the best way to estimate transfection efficiency. I found the most important factors to be transfection efficiency and integrity of the plasmids. Some reagents are more forgiving than others in grsnting consistent transfection even when some of the other factors may not be optimal (pH, the lot of the materials, etc.)