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Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein

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Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes.

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Data Availability

The data analyzed in this study are available from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge the Bioimaging platform of the Infectious Disease Research Centre, which is funded by an equipment and infrastructure grant from the Canadian Foundation for Innovation (CFI).

Funding

This work was supported by the Natural Sciences and Engineering Research Council (NSERC) and the Canadian Institute of Health Research (CIHR) (Collaborative Health Research Projects, CHRP program 315642).

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Authors and Affiliations

  1. Chemical Engineering Department, Université Laval, 1065, Avenue de la Médecine, Pavillon Pouliot, Québec, QC, G1V 0A6, Canada

    Juliette Champeil, Mathias Mangion & Bruno Gaillet

  2. PROTEO: The Quebec Network for Research on Protein Function, Structure, and Engineering, Université du Québec à Montréal, 201 Avenue du Président Kennedy, Montréal, QC, H2X 3Y7, Canada

    Juliette Champeil, Mathias Mangion & Bruno Gaillet

  3. ThéCell: FRQS Cell, Tissue and Gene Therapy Network, Laboratoire d’organogénèse expérimentale – LOEX, 1401, 18E rue, Québec, QC, G1J 1Z4, Canada

    Juliette Champeil, Mathias Mangion, Rénald Gilbert & Bruno Gaillet

  4. Human Health Therapeutics Research Center, National Research Council Canada, 6100, Avenue Royalmount, Montréal, Québec, H4P 2R2, Canada

    Rénald Gilbert

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Contributions

JC and BG conceived the idea of this study. Material preparation, data collection, and analysis were performed by JC. BG supervised the conduct of this study. The first draft of the manuscript was written by JC. RG and BG acquired the funding. All authors reviewed the manuscript draft and approved the final version for submission.

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Correspondence to Bruno Gaillet.

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Champeil, J., Mangion, M., Gilbert, R. et al. Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein. Mol Biotechnol 66, 1116–1131 (2024). https://doi.org/10.1007/s12033-023-01007-3

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  • DOI: https://doi.org/10.1007/s12033-023-01007-3

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