Fig 1.
Schematic workflow of the infectious titer assay performed with the Incucyte® S3.
HEK293T cells were seeded into a 96-well plate on day 1. The next day, the cells were transduced with diluted lentiviral vector and the transduction enhancer polybrene. 24 h post-infection the transduction mix was removed and replaced by a mixture of staining reagents containing FabFluor-488 labeled anti-FMC63 scFv antibody, Cytotox Red, and Opti-Green background suppressor. Dead cells were stained by Cytotox Red. Infected cells expressing the anti-CD19 CAR were stained by the FabFluor-488 labeled anti-FMC63 scFv antibody. Viable non-infected cells remained unstained. Quantification was performed by real-time fluorescence imaging from days 3-5.
Fig 2.
Workflow comparison of lentiviral vector (LV) infectious titer determination methods.
One day after cell seeding, cells are transduced with LV and transduction enhancer; measurement of cell number may be required to determine the cell number at time of transduction. On the third day the transduction mix is removed and replaced by growth medium and the staining mixture for the Incucyte® method. While the Incucyte®-based assay continually detects transduced cells with no further action required, the flow cytometry and RT-qPCR based assays require intensive handling for the final (endpoint) readout on day 4 or 5.
Fig 3.
Detection masks for infective titer calculation.
(A) Phase contrast image of HEK293T cells (left), merged with phase contrast area analysis mask in yellow (right). (B) HEK293T cells infected with lentiviral vector at a 1:625 dilution. The expressed CAR-construct was stained with an anti-FMC63 scFv antibody labeled with FabFluor-488. Phase contrast image merged with green fluorescence channel (left), background corrected green fluorescence channel (middle), and phase contrast image merged with green fluorescence analysis mask in magenta (right). (C) Live/dead staining positive control cells treated with 0.005% Triton X-100 and stained with Cytotox Red. Phase contrast image merged with red fluorescence channel (left), background corrected red fluorescence channel (middle), and phase contrast image merged with red fluorescence analysis mask in blue (right). All images were taken at 10x magnification.
Fig 4.
Time course series of normalized positive cell area.
(A) The FabFluor-488 positive area was normalized to the phase contrast area over time after transduction. The IgG1 isotype negative control and the lentiviral vector (LV) negative control (matrix) showed no FabFluor488 signal. The anti-transferrin-receptor IgG1 positive control antibody confirmed successful FabFluor488 labeling of the test antibodies. Matrix control overlaps with the negative control. Data represent the mean ± standard deviation of three technical replicates. (B) Normalized FabFluor488 peak value vs. time at which this value is reached. The datapoint in the upper left with the highest normalized positive area represents the 1:2 LV dilution. The other datapoints represent two-fold serially diluted LV up to a dilution of 1:1024 with the lowest normalized positive area and the longest time after which the peak value is reached. Data represent the mean ± standard deviation of three biological replicates.
Fig 5.
Linear range of the infectious titer assays.
The number of positive cells detected via flow cytometry (A & B) and the FabFluor-488 positive area determined with the Incucyte® S3 (C & D) were plotted against dilution factors from a serially diluted lentiviral vector. A logarithmic trend was observed across the whole range of dilutions using the Incucyte® readout with an R2 of 0.98 (C) and the flow cytometer readout with an R2 of 0.95 (A). A linear dependence was determined for dilutions from 1:64 to 1:512 for the Incucyte® assay with an R2 of 0.96 (D) and from 1:128 to 1:512 for the flow cytometer assay with an R2 of 0.988 (B). Data represent the mean ± standard deviation of three biological replicates.
Table 1.
Inter-assay and intra-assay precision of the Incucyte® infectious titer assay.
Fig 6.
(A) Correlation of cell count and cell confluence. The cell confluence measured 7 h after cell seeding with the Incucyte® was plotted against the cell count seeded per well as determined by the Cedex HiRes® Analyzer. Mean ± standard deviation, linear regression fit applied (red line) with R2 = 0.99. (B) Infective titer for viable cells determined by the Incucyte® S3 and by flow cytometry. (C) Infective titer for three samples of different virus dilutions within the linear range of the Incucyte®-based assay calculated either for all cells or for viable cells. Data represent the mean ± standard deviation of three biological replicates.
Fig 7.
Investigation of external factors on lentiviral vector stability.
Assessment of the influence of repeated freeze and thaw cycles (A), storage at different temperatures over time (B), control line represents non-treated LV sample, different sodium chloride (NaCl) concentrations (C) and shear stress induced by a peristaltic pump (D) on lentiviral vector (LV) infectivity. Experiments were performed in biological triplicates. Data represent the mean ± standard deviation. P-values are indicated as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.