Application Note: Real-Time Live-Cell Analysis of Lentiviral Titer Determination
Authors: Kalpana Barnes, Jennifer J. Labisch, Philip Wiese, Karl Pflanz | Last Updated: January 2022
Overview
Viral vectors are pivotal in gene therapy, with lentiviral vectors (LV) being the primary choice for CAR-T cell therapies. However, the quality control of these vectors is a significant bottleneck in process development and production. Traditional methods like qPCR and flow cytometry are limited by low throughput and extensive manual handling.
To overcome these challenges, we introduce a novel, real-time, image-based approach using the Incucyte® Live-Cell Analysis System. This method leverages Incucyte® Fabfluor-488 Antibody Labeling and Incucyte® Cytotoxicity Dyes for kinetic quantification of functional lentiviral titers in live cells. This innovative solution addresses the need for rapid and accurate infectious titer determinations, facilitating both process development and optimization.
- Document type: Application Note
- Page count: 9
- Read time: 16 minutes
Key Takeaways
- A simple, rapid, non-invasive approach for high-throughput functional quantification of infectious titers
- How real-time, kinetic readouts and integrated software enable viable transduced cell tracking over time
- Exploration of real-time titer insights, in-process monitoring, and lentiviral vector production optimizations
- Assay flexibility and applicability to determine optimal processing conditions for improved viral vector development
Incucyte® Lentivirus Titer Assay Workflow
Figure 1. HEK293T cells were seeded (6 x 103 cells, 50 μL per well) into black walled 96-well poly-L-lysine coated tissue culture treated plates (Corning). On day 2, cells were transduced with a dilution series of lentiviral vector in the presence of polybrene (8 μg/mL). 24 h post-transduction, transduction mix was removed and replaced with a cocktail of detection reagents (50 μL, 2.5 μg/mL FAC Incucyte® Fabfluor-488 labeled anti-FMC63 scFv antibody 1:200 dilution, 50 μL, 0.5 mM FAC Incucyte® Opti-Green background suppressor, 50 μL, 250 nM FAC Incucyte® Cytotox Red).
