Hi everyone
I produced a batch of virus , enriched it with centrifugation, and titrated it with T cells after 48h. For my experiments I want to use MOI 3 and that's equal to 1/12 dilution.
but at this dilution my cells will die after 5 days in cell culture( I use PBMC not sorted T cells), while untraduced control expanded 6 times during 5 days of stimulation. Until day 3 transduced cells are similar as untraduced cells. I tried up to 1/160 dilution but I still have very large cell death with this dilution!
Do you have any idea what is the reason for this?
Thanks
Hello Reza,
May I ask for some more details:
Is untransduced control handled same way as your transduced experimental arms?
Do all experimental Arms ( including untransduced control) have same type of Media?
What is the % of FBS in your transduction Media? It should be same in your untransduced control arm, for fair comparison.
Transduction time
If cell death is truly caused by viral activity, even at such low titer as 1/160 dilution, I would try and decrease transduction time.
Change to fresh Media
If pronounced viral toxicity is observed, you have the option to change to fresh media after 24 hrs of transduction and see if that makes difference.
Seeding Density of PBMC
you can also try and increase seeding density of PBMC.
Best,
Nino
Hi Nino
Thanks for your answer,
the media is the same and I'm sure it's virus toxicity, in my protocol I don't change the medium until it's necessary ( for adjusting the cell concentration or too much acidity). I start at 106/ml cell density and every time I add the media I try to keep it at 0.8 million/ml.
I tried with 1/320 dilution ( I enriched the virus with ultracentrifugation) and I still had large cell death in the medium and good transduction at day 7( I attached flow graphs at day 7, my CAR construct also has a GFP marker ). Do you think it is better to change the medium after 24h or reduce virus even more?
thanks
Thank you for additional info Reza.
Decreasing viral titer from 1/160 to 1/320 does not seem to help your Lymphocyte count and transduction efficiency drops from 56% to 40%.
It will be interesting to see if diluting further will help. However, I am suspecting that your transduction efficiency will drop as well.
What is the end goal of your experiment? Are you trying to establish T cell population with CAR construct that can be used for future experiments/assays?
If you have access to FACS, maybe you can sort for transduced population and try and expand it.
Your transduction efficiency is highest at 1/40 dilution. Another approach would be to transduce at even higher titer( your transduction efficiency should increase at least until your cells start to die), observe until cells start to die, check transduction efficiency, sort and expand.
I would definitely try and change medium after 24 hrs and see if that helps.
Transductions are tricky. Lots of optimization needed until you get to finish line.