HEK293T cells were used as the lentivirus stable transfection packaging cells. After transfected my target cells with the lentivirus (included puromycin resistance)
produced, I got a colony of cells with much higher proliferation rate than the rest of the colonies after puromycin selection and somewhat different cytomorphology from that of other colonies too. I used 1250 rpm centrifuge for 5 minutes to spin down and get rid of any possible HEK293T cells from the HEK293T culture media containing lentivirus particles and used the supernatant to transduce my target cells. The supernatant containing lentivirus particles was also stored in the 4 degree refrigerator overnight before my lentivirus transduction of target cells the next day. Could it still be a possibility that a HEK293 T cell contaminated the media containing the lentivirus particles, survived 24 hours in a 4 degree environment without CO2 and was transfected by the lentivirus like the target cells?
No, it should not be an issue. The centrifugation step should pellet all the cells.
If you are still unsure, you can try the following:
1. 3000 rpm for 15 minutes at 4°C to pellet cell debris instead of 5 minutes,
2. storage at -80'C, but avoid freeze thaw cycles,
3. Filtering is not recommended, but if you must filter out contaminants, use a low-protein binding 0.45 micron filter or a larger pore-size.
I usually use this protocol for packaging. It might be useful for you.
https://info.abmgood.com/lentivirus-system-production-protocol#packaging
Most cases it will not Be a problem if the supernatant is filtered before use. The exosomes could still be there in centrifuged supernatants and has a possibility of introducing nucleic acids in target cells. It is good to go for a DNA fingerprinting of target cells after getting stable cells to be sure.
Thank you for all the answers. I did not do the filtering, only centrifuge and then was store in 4 degree refrigerator before used the supernatant for lentivirus transduction. what kind of filter is low protein binding? Will 0.45um PES filter work?
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