An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids

When the spheroids reach the appropriate experimental size, carefully angle the plate, and use a multichannel pipette to gently remove 150 microliters of complete medium from each well without disturbing the spheroids. Next, replace the discarded medium with 50 microliters of a 1-to-200 solution of Annexin-V red, and place the plate in a cell culture incubator for 15 minutes.

Centrifuge transduced CAR CD19 T cells in a 15-milliliter conical tube, and resuspend the pellet in 2 milliliters of complete medium after counting, dilute the cells to a 2 x 10⁵ cells per milliliter of complete medium concentration.

Then, add 100 microliters of CAR CD19 T cells to each spheroid containing well, and return the plate to the automated imaging apparatus. In the acquisition software, select Schedule to Acquire and right-click on the Scan Timeline. Select Edit Timeline, and right-click on the Scan Group to delete it.

Right-click on the timeline again, and select Set Selected Scan Group Interval, and Set Add Scans Every to 1 and 1/2 hours and for a total of to 24 hours. Then set the imaging start time to at least one hour after the start of the incubation in the automated imaging apparatus, and select Save Scheduled Scans.