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

1. 3D Tumor Spheroid Killing Assay

1. After 6 days or once spheroids reach the desired size, remove the plate from the incubator. Using a multichannel pipette, gently remove 100 µL/well of complete RMPI 1640 medium from the spheroid plates.​
   1. For this step, angle the tips towards the inside wall of the 96-well plate, avoiding contact with the bottom of the well in order to minimize disturbance of the spheroids. The remaining volume should be around 100 µL.
2. Prepare a 1:200 solution of Annexin V red by mixing 50 µL of Annexin V red with 9.95 mL of complete RPMI 1640 medium.
3. Add 100 µL/well of the 1:200 Annexin V red solution.
4. Transfer the plate to an incubator (37°C, 5% CO₂, 95% humidity) for 15 min.
5. Harvest the transduced CAR CD19 T cells in a 15 mL tube and centrifuge them at 500 x g for 5 min. Remove the supernatant using a pipette and resuspend by pipetting up and down several times with 2 mL of complete RPM1 1640 medium.
6. Count cells using Trypan blue exclusion on a compatible cell counter.
7. Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend in RPMI medium to obtain 2 x 10⁵ cells/mL.
8. Transfer the cell suspension to a sterile reservoir and dispense 100 µL/well into a 96-well round bottom spheroid plate using a multichannel pipette.
9. Transfer the plate back to the automated imaging apparatus inside an incubator (37 °C, 5% CO₂, 95% humidity).
10. Log into the acquisition software,Select Schedule To Acquire.
    1. Right-click on the Scan timeline and select Edit Timeline. Right-click on the scan group and delete it. Right-click on the timeline and select Set Selected Scan Group Interval option and set Add scans every to 1.5 h and For a total of to 24 h.
    2. Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus). Select Save schedule scans.