1. Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.
- Author
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Velikonja C, Steenbakkers L, How J, Enns M, Corbett B, McCready C, Nease J, Mhaskar P, and Latulippe D
- Subjects
- Humans, Viral Load methods, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Algorithms, Adenoviridae genetics, Adenoviridae isolation & purification
- Abstract
Interest in virus-based therapeutics for the treatment of genetic and oncolytic diseases has created a demand for high-yield, low-cost virus-manufacturing processes. However, traditional analytical methods of assessing infectious virus titer require multiple processing steps and manual counting, limiting sample throughput, and increasing human error. This bottleneck severely limits the development of new manufacturing unit operations to drive down costs. In this work, we utilize an Incucyte Live-Cell Analysis System to develop a high-throughput infectious titer assay for adenovirus expressing a GFP-transgene. Although previous studies have demonstrated live-cell imaging's potential for use with other viruses, they provide little guidance regarding the selection of the viewing and analysis parameters. To fill this gap, we develop an algorithmic approach to identify the optimum viewing and analysis parameters and create a statistical workflow for quantifying infectious adenovirus in a sample dilution series in a standard 24-well microplate. The developed assay is comparable to Hexon staining, the gold-standard for adenovirus infectious titer, with a Pearson correlation coefficient of 0.9. Finally, the developed algorithmic approach and statistical workflow were applied to create an assay for adenovirus titer using a 96-well microplate, allowing five times more samples to be quantified compared to the standard 24-well plate. While this assay uses a GFP-insert that precludes its use in a clinical environment, the key learnings surrounding the careful use of viewing and analysis parameters, and the statistical workflow are widely applicable to implementing life-cell imaging for dilution-series-based assays. Moreover, this method directly enables the fast and accurate evaluation of virus samples in a preclinical environment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Latulippe reports financial support was provided by Mitacs. David Latulippe reports financial support and equipment, drugs, or supplies were provided by Sartorius Corporation. Landon Steenbakkers reports financial support was provided by BioCanRx. Joshua How reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Claire Velikonja reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Brandon Corbett reports a relationship with Sartorius Corporation that includes: employment. Chris McCready reports a relationship with Sartorius Corporation that includes: employment. Prashant Mhaskar reports a relationship with Sartorius Corporation that includes: funding grants. David Latulippe reports a relationship with Sartorius Corporation that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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