A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

For a real-time cytolysis potency assay, add 50 to 100 microliters of target cell culture medium to each well of an E-plate, and measure the background impedance in the cell-analyzer instrument.

Detach the target cancer cells from the culture plate using trypsin. Wash the cells in 15 milliliters of fresh culture medium and centrifuge. Re-suspend the cancer cell pellet in 5 milliliters of fresh medium for counting and adjust the cell density to the appropriate target cell concentration. Add the cells to the appropriate number of wells, and leave the E-plate for 30 minutes at room temperature to allow the cells to settle evenly on the bottom of the wells.

At the end of the equilibration period, place the plate in the real-time cell-analyzer, and initiate measurement of the impedance automatically every 15 minutes overnight. The adhesion and proliferation of the target cells can be tracked in real-time.

The next morning, dilute the effector CAR and control T cells to the appropriate concentration in individual tubes, and remove 50 to 100 microliters from each well of the E-plate so that the final volume in each well is 100 microliters. Next, see the effector and control T cells in the appropriate wells at the appropriate effector-to-target ratios in 100 microliters of medium per well. Then, equilibrate the plate for 30 minutes at room temperature before returning the plate to the cell-analyzer system.

To measure the efficacy of the T cell-mediated killing of the target cancer cells, monitor the CAR-T cell-mediated killing for 96 hours.