Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production.
Journal
Research (Washington, D.C.)
ISSN: 2639-5274
Titre abrégé: Research (Wash D C)
Pays: United States
ID NLM: 101747148
Informations de publication
Date de publication:
2020
2020
Historique:
received:
09
08
2018
accepted:
28
01
2020
entrez:
8
4
2020
pubmed:
8
4
2020
medline:
8
4
2020
Statut:
epublish
Résumé
The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.
Identifiants
pubmed: 32259105
doi: 10.34133/2020/1387402
pmc: PMC7094759
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1387402Informations de copyright
Copyright © 2020 Daniel J. Blackstock et al.
Déclaration de conflit d'intérêts
The authors declare that there is no conflict of interest regarding the publication of this article.
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