eBook: Cell and Gene Therapy - 13

Figure 4. CD20 CAR-T in vitro killing assay of Daudi-Luc2 measured using luminescence and live cell imaging. (A) CD20-positive Daudi- Luc2
cells (5 x 103
) were seeded into a 96-well plate and were used as target cells for either CD20 CAR-T or Mock CAR-T (control) from the same donor,
which were seeded at various ratios of CAR-T cells to target Daudi-Luc2 cells (1:1, 2:1, 5:1, and 10:1). After 24 hours of co-culture, Bright-Glo was
added to the indicated wells. The luminescence of the plate was read within 10 minutes using a luminescence plate reader and determined to
have a dose-dependent-specific killing with CD20 CAR-T cells, which was greater than the non-specific killing observed with mock CAR-T cells (*
= significant difference and ns = not significant using unpaired t test, with a single pooled variance). (B) Daudi-Luc2 cells (5 x 103
) were co-cultured
with CD20 CAR-T cells or Mock CAR-T cells in the presence of Incucyte Cytotox red dye in the medium and real-time fluorescent imaging
was measured every hour for 24 hours, resulting in an increase of fluorescence intensity when co-cultured with CD20 CAR-T as compared to
co-cultures with Mock-CAR-T cells. (C) After 24 hours of co-culture with CD20 CAR-T cells, Daudi-Luc2 showed an increase in the number dead
(red) fluorescent cells as compared co-culture with Mock CAR-T cells. (D) The clustered red fluorescence was quantified and compared (* = significant
difference and ns = not significant using unpaired t test, with a single pooled variance).
with HER2 CAR-T cells show a greater decrease
in cell impedance as the cells lift off the plate as
compared to co-culturing with mock CAR-T cells
or BT-474-Luc2 cells alone.
Conclusions
The CAR-T Target Luciferase Reporter Cells described
in this study provide an advantage in mea13
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