Abstract
Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor-and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.
Original language | English |
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Article number | e66532 |
Journal | Journal of Visualized Experiments |
Volume | 2024 |
Issue number | 206 |
Early online date | 26 Apr 2024 |
DOIs | |
Publication status | Published - Apr 2024 |
Bibliographical note
Publisher Copyright:© 2024 JoVE Journal of Visualized Experiments.
Funding
This work was supported by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) research fund and a grant from Start2Cure (0-TI-01). We thank Leisha Kopp for her input and advice in the setup of the protocol. Figures were created using Biorender.
Funders | Funder number |
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Koninklijke Nederlandse Akademie van Wetenschappen | 0-TI-01 |
Koninklijke Nederlandse Akademie van Wetenschappen |