Lentivector supe collection

Most lentiviral production protocols (usually with VSV-G pseudotyped particles) tells the user to collect the supe at 48 or 72 hours. My protocols tend to say collect the supe twice a day (once when coming into the lab, and once when leaving) starting at 24 hours and ending a few days later (96 hours? more?).

This largely stems from a point in my PhD when I was generating a bunch of VSV-G pseudotyped lentiviral particles to study the “early stage” of the HIV life cycle (ie. the points preceding integration into the genome, such as the trafficking steps to get into the nucleus). After thinking about the protocol a bit, I realized that there’s really nothing stopping the produced VSV-G pseudotyped particles from attaching and re-entering the cells they emerged from, which is useless for viral production purposes. Even for the particles that are lucky enough not to re-enter the producer cells, they are going to be more stabler in a less energy environment (such as 4*C) than floating around in the supe at 37*C in the incubator.

But, well, data is always better to back such ideas. So back in April 2014 (I know this since I incorporated the date into the resulting data file name), I did an experiment where I produced VSV-G pseudotyped lentiviral particles as normal, and collected the supe at ~12 hour intervals, keeping them separate in the fridge. After they were all collected, I took ~ 10uL from each collected supe, put them on target cells, and measured luciferase activity a couple of days later (these particles had a lentiviral vector genome encoding firefly luciferase). Here’s the resulting data.

Some observations:

  • Particles definitely being produced by 24 hours, and seemingly reaching a peak production rate between 24 and 48 hours.
  • The producer cells kept producing particles at a reasonable constant rate. Sure, there was some loss between 48 and 72 hours, but still a ton being produced.
  • I stopped this experiment at 67 hours, but one can imagine extrapolating that curve out, and presumably there’s still ample production happening after 72 hours.

So yea, I suppose if the goal is to have the highest singular concentration, then taking a single collection at 48 or 72 hours will probably give you that. That said, if the goal is to have the highest total yield (which is usually the situation I’m in), then it makes much more sense to collect at various intervals, and then use the filtered, pooled supe in downstream experiments.

Also, I consider being able to dig up and discuss 10-year old data as a win!