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

Autor: Champeil J; Chemical Engineering Department, Université Laval, 1065, Avenue de la Médecine, Pavillon Pouliot, Québec, QC, G1V 0A6, Canada.; 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.; ThéCell: FRQS Cell, Tissue and Gene Therapy Network, Laboratoire d'organogénèse expérimentale - LOEX, 1401, 18E rue, Québec, QC, G1J 1Z4, Canada., Mangion M; Chemical Engineering Department, Université Laval, 1065, Avenue de la Médecine, Pavillon Pouliot, Québec, QC, G1V 0A6, Canada.; 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.; ThéCell: FRQS Cell, Tissue and Gene Therapy Network, Laboratoire d'organogénèse expérimentale - LOEX, 1401, 18E rue, Québec, QC, G1J 1Z4, Canada., Gilbert R; ThéCell: FRQS Cell, Tissue and Gene Therapy Network, Laboratoire d'organogénèse expérimentale - LOEX, 1401, 18E rue, Québec, QC, G1J 1Z4, Canada.; Human Health Therapeutics Research Center, National Research Council Canada, 6100, Avenue Royalmount, Montréal, Québec, H4P 2R2, Canada., Gaillet B; Chemical Engineering Department, Université Laval, 1065, Avenue de la Médecine, Pavillon Pouliot, Québec, QC, G1V 0A6, Canada. bruno.gaillet@gch.ulaval.ca.; 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. bruno.gaillet@gch.ulaval.ca.; ThéCell: FRQS Cell, Tissue and Gene Therapy Network, Laboratoire d'organogénèse expérimentale - LOEX, 1401, 18E rue, Québec, QC, G1J 1Z4, Canada. bruno.gaillet@gch.ulaval.ca.
Jazyk: angličtina
Zdroj: Molecular biotechnology [Mol Biotechnol] 2024 May; Vol. 66 (5), pp. 1116-1131. Date of Electronic Publication: 2024 Jan 05.
DOI: 10.1007/s12033-023-01007-3
Abstrakt: 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 × 10 6  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.
(© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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