eBook: Cell and Gene Therapy - 12

various target to effector ratios (1:1, 2:1, 5:1, and
10:1). After co-culturing for 24 hours, the BrightGlo™
Luciferase Assay System (Promega) was used
and luminescence was measured on a plate reader.
Analysis showed a clear dose-dependent decrease
in luminescence, indicating the antigen-specific
CAR-T killing potential was greater
than the non-specific killing observed when
co-culturing with mock CAR-T cells (Figures 2A,
2B, 3A, 4A, and 5A). Live cell imaging studies
were conducted using the same method and
were observed microscopically every 30 minutes
to 1 hour using the Cytation 1 system (Agilent).
The luciferase-expressing cells were either prestained
with Vybrant™ (Thermo Fisher) DiO (Figure
2C and 2D) or co-cultured in the presence of
Incucyte® Cytotox red dye (Sartorius)5
(Figures
4B and 5B). Incucyte Cytotox red, which stains
dead cells, shows a dose-dependent increase in
fluorescence intensity when cells are co-cultured
with antigen-specific CAR-T cells (Figures 4C, 4D,
5C, and 5D). BT-474-Luc2 when co-cultured with
HER2 CAR-T cells or mock CAR-T cells from the
same donor were measured in a real-time cell
analysis assay using the xCelligence (Agilent)6
(Figure 3B) system and impedance was measured
every 15 minutes. BT-474-Luc2 cells co-cultured
Figure 3. HER2 CAR-T in vitro killing assay of BT-474-Luc2 measured using luminescence assay and xCelligence. (A) HER2-positive BT-474- Luc2
cells (5 x 103
) were seeded into a 96-well plate and were used as target cells for either HER2 CAR-T or Mock CAR-T (control) from the same donor,
which were seeded at various ratios of CAR-T cells to target BT-474-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 HER2 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) HER2 CAR-T cells were used to target
2 x 104
HER2-positive BT-474-Luc2 at a 10:1 ratio and cell killing was measured using the xCELLigence system. Mock CAR-T cells from the same
donor were used as a control.
12
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