Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

Autor:	Labisch JJ; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany.; Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Lower Saxony, Germany., Wiese GP; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany.; Faculty of Mathematics, Computer Science and Natural Sciences, RWTH Aachen University, Aachen, North Rhine-Westphalia, Germany., Barnes K; BioAnalytics Applications, Essen BioScience, Royston, Hertfordshire, United Kingdom., Bollmann F; Segment Marketing Viral-based Therapeutics, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany., Pflanz K; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany.
Jazyk:	angličtina
Zdroj:	PloS one [PLoS One] 2021 Jul 15; Vol. 16 (7), pp. e0254739. Date of Electronic Publication: 2021 Jul 15 (Print Publication: 2021).
DOI:	10.1371/journal.pone.0254739
Abstrakt:	The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity. Competing Interests: The authors declare no competing financial, professional, or personal interests that may have influenced the work described in this manuscript. The commercial affiliation reported in the financial disclosure statement did not give rise to any type of competing interest. The commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.
Databáze:	MEDLINE
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