Optimization of viral vector production in suspension cells using plasmid transfection method
Sinkkonen, Riku (2018)
2018
Teknis-luonnontieteellinen
Teknis-luonnontieteellinen tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2018-12-05
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201811192616
https://urn.fi/URN:NBN:fi:tty-201811192616
Tiivistelmä
The aim of this study was to develop a protocol to produce AAV (adeno-associated virus) in suspension cells using plasmid transfection method. This is because suspension cell culture is easier to scale-up than adherent cell culture. The aim of this study was to get the AAV yield with suspension cells as high as with adherent cells. This study was done at KCT (Kuopio Center for Gene and Cell Therapy Oy).
Different transfection reagents, transfection efficiency enhancers, cell culture and transfection media, transfection mixture incubation times, reagent ratios, cell densities and other parameters were tested to find the best protocol to get as high transfection efficiency and viral titer as possible. Over 200 experiments were performed to find the final protocol. The transfection efficiency was measured using flow cytometry, and the viral titer was analyzed using qPCR (quantified PCR).
Cell line used was 293T, and these experiments were carried out in mini bioreactor tubes. Transfection efficiencies of over 95 % and viral titer of over 7*1011 vg/mL were achieved in 10 mL scale. Results were obtained using cell culture media Expi293 and LV-MAX, and transfection reagent PEI (polyethylenimine). Optimal conditions regarding DNA:PEI ratio, incubation time, and cell density were determined. This was also tested in 50 mL scale using roller bottles. These gave only around 30 % transfection efficiency, but the viral titer was over 1*1012 vg/mL. These results obtained with suspension cells were on the same level as achieved earlier at KCT with adherent cells.
Different transfection reagents, transfection efficiency enhancers, cell culture and transfection media, transfection mixture incubation times, reagent ratios, cell densities and other parameters were tested to find the best protocol to get as high transfection efficiency and viral titer as possible. Over 200 experiments were performed to find the final protocol. The transfection efficiency was measured using flow cytometry, and the viral titer was analyzed using qPCR (quantified PCR).
Cell line used was 293T, and these experiments were carried out in mini bioreactor tubes. Transfection efficiencies of over 95 % and viral titer of over 7*1011 vg/mL were achieved in 10 mL scale. Results were obtained using cell culture media Expi293 and LV-MAX, and transfection reagent PEI (polyethylenimine). Optimal conditions regarding DNA:PEI ratio, incubation time, and cell density were determined. This was also tested in 50 mL scale using roller bottles. These gave only around 30 % transfection efficiency, but the viral titer was over 1*1012 vg/mL. These results obtained with suspension cells were on the same level as achieved earlier at KCT with adherent cells.