We are producing Lentiviral Vectors with CaPO4 transfection. And trying to optimize our protocol. There are two steps that we couldn't decide. One of them is the speed and degree of ultracentrifugation. We use 19500 rpm with SW28 rotor at 4 C. There are different parameters in literature like 25000 rpm, and 16-20 C. Is there any specific effect on viral particle titer? Another challenge is about resuspension of particles after centrifugation. We have tried overnight resuspension with 1X PBS , at +4 C. And our concentration fold was 350X. But there are different suggestions like incubating at room temperature for 1 hour on a rotator incubator. Or just pipetting, collecting and a quick pulse with a micro centrifuge. Which way would be more effective and how fold should we concentrate our lentiviruses? I would be appreciate for any recommendations.
All the variants of the method you describe are fine and result in the same titers.
We pipet normally 20-60 times (avoiding bubbles!!), incubate then for about an hour on a rotator incubator at 4°C and some in our group include some short pulses on the vortex. The fold of concentration just depends on the amount of resuspension medium (down to 30µl per SW28 tube are possible). More important for good titers is the efficiency of transfection.
But what really matters to keep the titers is fast freezing and thawing, if you store them at -80°C.
All the best,
Irene
Hi Hale, I just read your question and I felt like myself asking the same question few weeks ago. I am currently optimizing a protocol for LV production. As Irene said, for a good titer it is very importat the efficiency of your transfection. How much do you need? that depends on what you will apply your LV. Some people just freeze the supernatant 48-60 hours post -transfection for in-vitro assays and works very well (I did it). But, if you want to inyect your virus in a animal model, off-course you will need it more concentrated and purified.
Do you know your transfection efficiency? I use to do it by FACS 48-60hrs after transfection. For the purification, first I filtrate the supernatant with 0.45uM filter and ultracentrifuge (50 000g) with SW40ti rotor for 90 min at 4C. Then, I dissolve the pellet (for me invisible) with 5 ml PBS (between 3-4 hours on ice). I got 150X best results. But as I said before, the amount of virus will depend on what do you want it for.
Thanks for both answers . We will use our viral vectors both in vivo and in vitro experiments. So we are trying to produce more concentrated and purified vector as possible. We are now considering to use some reagents for transfection efficiency. But still we want to be sure about if we can resuspend and collect all particles that we produce. The most difficult step for us is pipetting with 50-100 ul PBS for SW28 tubes without bubbles!! I have read that bubbles have negative effect on virus titer.
Yes, this is the task... :-)
But one suggestion: if you use e.g. 50µl for resuspension adjust the pipet to 30µl for pipetting up and down, then you avoid the accumulation of bubbles as they are really bad.
- Badges
- Science method