eBook: Cell and Gene Therapy - 14

suring target cell killing without the use of a radioactive
51
for CAR-T functional evaluation.4
reporter cell lines were characterized and authenCr
release assay or pre-labeling the cells
In addition, these
ticated using cell morphology, growth kinetics,
and STR analysis.7
The expression stability of both
the target antigen and luciferase was verified by
comparing the low-passage and the high-passage
Figure 5. CD20 CAR-T in vitro killing assay of Farage-Luc2 measured using luminescence and live cell imaging. (A) CD20-positive Farage- 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 Farage-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) Farage-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, Farage-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).
14
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