In Vitro Assessment of Cytotoxicity Mediated by Hypoxia-Sensitive CAR-T Cells

To begin, seed 1000 target cells into each well of two flat bottom 96 well tissue culture plates containing 200 microliters of FBSDMEM. The next day carefully pipette out 100 microliters of the supernatant from the top of each well. Add chimeric antigen receptor T cells or nontransduced T cells in 100 microliters of FBSDMEM at different ratios.

Place one plate in a 21%oxygen atmosphere and the other in a mobile carbon dioxide oxygen nitrogen incubator chamber under 1%oxygen atmosphere. After 24 hours of co-culturing, use a pipette to carefully transfer all the supernatant into a new U-bottom 96 well plate. Store the supernatant at minus 20 degrees Celsius for cytokine detection.

Now add 60 microliters of passive lysis buffer into each experimental well of the black flat-bottom 96 well plates. Place the plates on a shaker for 30 minutes to ensure efficient cell lysis. Add 60 microliters of firefly luciferase substrate into each experimental well.

Use a microplate reader to measure the luciferase activity immediately. Finally, calculate the normalized cytotoxicity percentage with the given equation. The hypoxia sensitive HER2 targeting CAR was able to effectively kill target cells regardless of whether the atmosphere was hypoxic or normoxic.

In contrast, the HER2-BBz-ODD CAR T cells displayed significantly weaker cytotoxicity under normoxic conditions for all conditions.