Hi everybody, I'm trying to reprogram PBMCs isolated from peripheral blood to iPSCs using Cytotune 2.0 kit. I've tried to do it feeder-free using Matrigel for coating and ReproTeSR (StemCell technologies) but it doesn't work. :(
I've done several attempts with different MOI (5:5:5 and 5:5:3) and also using Polybrene with any success. I'm desperated!
Has anybody any tips about this?
Lots of thanks
Polybrene helps with lentiviral transductions, but I don't believe it has any benefit for Sendai virus.
Thanks for your answer! I decided to use Polybrene because in the users guide of Cytotune says that Polybrene improves the efficiency...
OK, that must be a relatively new addition. I certainly don't recall that in the original version. I highly doubt it will hurt to add it.
When you say that the reprogramming does not work, could you please clarify how it doesn't work? Also, how are you doing your matrigel coating? I ask because a lot of people do not seem to understand how to do proper matrigel handling and coating.
Ron Hrstka Thanks for your time!
When I said that reprogramming doesn't work I mean that I haven't seen any colony formation (based on my experience in fibroblast reprogramming).
The only things I saw were a few aggregates of cells that disappeared the next few days with the medium changes and single cells attached to the plate.
30 days after transduction I can’t see any sign of reprogrammed cells, the only adhered aggregates in the plate doesn’t change their morphology and furthermore, they do not grow.
I perform Matrigel coating always with cold tips and pipettes. I have aliquots (as the dilution factor in the data sheet of Matrigel says) and prior to use it, I dilute one aliquot in DMEM/F12 on ice. I coat the plates with the mixture of DMEM/F12 and Matrigel and keep it at room temperature for 1 hour before use it.
I was hoping the problem was "simple," but if you have experience with fibroblast reprogramming then you already have those potential basic issues sorted out. Good luck.
Hi Victoria! Polybrene is only recommended if you are not transducing via centrifugation. If both are employed simultaneously, depending on the cell that you expand from the pool of PBMCs, there'll be excessive cell death leading to failed reprogramming. Have you tried polybrene alone, or centrifugation alone? It is not recommended to perform both on the same sample.
Hi Victoria Cerrada López . Have you fixed your problem? I'm having exactly the same problems as you. I'm struggling with my iPSC generation for more than 6 months and I'm still not able to generate them. I have tried with matrigel/ irrMEF/irrHFF/MMC-HFF and no colonies were obtained at the end.
Hi Victoria,
The cytotune kit reccomends Vitronectin for feeder free PBMC transductions, I have found this to work, Surebond XF however was better.
Best of Luck!
Rose
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