Repetitive Tumor Challenge Assay: An In Vitro Culture Assay to Evaluate Chimeric Antigen Receptor (CAR) T Cells for Repetitive Tumor Killing Potential

Begin by dissociating glioblastoma tumor spheres from a glioblastoma tumor sphere culture with 1 milliliter of cold accutase and 30 to 60 seconds of pipetting. When the tumor spheres have been disrupted, stop the reaction with 5 milliliters of warm co-culture medium and pellet the tumor cell suspensions by centrifugation.

Resuspend the glioblastoma cells to a 1.6 times 10 to the fifth tumor cells per milliliter of fresh co-culture medium concentration and dilute T cells harvested from a CAR T cell culture to the appropriate percentage of CAR positive T cells per milliliter of co-culture medium concentration.

Next, add 100 microliters of the diluted tumor cells to each well of a 96-well flat-bottom tissue culture plate and 100 microliters of diluted CAR T cells into each well of tumor cells with gentle mixing. Then, place the plate in a 37 degrees Celsius, 5% carbon dioxide incubator.

On days 2, 4, and 6 of culture, carefully remove 50 microliters of medium from each well of the tumor cell T cell co-culture plate slated for rechallenge according to the table. Then, add 50 microliters of fresh glioblastoma cell suspension prepared as just demonstrated, but with twice the cell number of the initial co-culture to each well with gentle mixing, and return the plate to the cell culture incubator.

Days 1, 3, 5, and 7 of co-culture, transfer the supernatant from each well to be harvested into a new 96-well round-bottom plate according to the table and add 50 microliters of pre-warmed 0.5% trypsin-EDTA into each medium depleted well. After 5 minutes at 37 degrees Celsius, confirm that the cells have detached from the bottom of each well by light microscopy and gently pipette the enzyme solution around the bottom of each well to resuspend the cells before transferring the detached cell suspension into the appropriate corresponding wells of the new 96-well plate.

Pellet the cells by centrifugation and wash the cells with 200 microlitres of fluorescence-activated cell sorting staining solution, or FSS, per well with a second centrifugation. Resuspend the pellets in 100 microliters of the antibody cocktail of interest in 100 microliters of FSS per well for a 30-minute incubation at 4 degrees Celsius.

At the end of the incubation, remove any unbound antibody by sequential 100 and 200 microliter FSS washes and resuspend the cells in 100 to 200 microlitres of DAPI nuclear stain and FSS. Then, analyze the cells according to standard flow cytometric analysis protocols.