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

1. Media Preparation

1. Prepare neural stem cell media for culturing primary GBM cell lines: DMEM:F12, 1:50 B27, 5 µg/mL heparin, and 2 mmol/L L-glutamine; supplemented with 20 ng/mL epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (FGF) twice a week (see Table of Materials).
2. Prepare T cell media: X-VIVO 15 containing 10% fetal calf serum (FCS); supplemented with 70 IU/mL rhIL-2 and 0.5 ng/mL rhIL-15 every 48 h (see Table of Materials).
3. Prepare co-culture media: take neural stem cell media without EGF and FGF supplement, and add 10% FCS.
4. Prepare FACS staining solution (FSS): HBSS, 2% FCS, NaN₃ (0.5 g/500 mL).

2. Preparation of GBM Tumor Cells

1. Harvest low-passage GBM tumor spheres (TSs) by centrifugation at 300 x g for 4 min and discard supernatant.
   NOTE: GBM tumor spheres (TSs) are generated from resected tumors as described previously, and maintained in neural stem cell media, in incubators with 5% CO₂ at 37 °C.
2. Pre-warm co-culture media in a 37 °C water bath.
3. Add 1 mL of cold accutase to GBM TSs, dissociate TSs by pipetting for 30–60 s, and stop dissociation by adding 5 mL of warm co-culture media.
   NOTE: GBM TSs should not be kept on accutase for more than 5 min.
4. Harvest GBM cells by centrifugation at 300 x g for 4 min, discard supernatant, and resuspend cells in 2 mL of co-culture media.
5. Determine cell concentration using a cell viability counter.
   NOTE: Cell viability must be >70%.

3. Preparation of CAR T Cells

1. Less than 24 h before the assay, take 100 µL of cultured CAR T cells into a flow cytometry tube and add 2 mL of FSS.
   NOTE: CAR T cells were generated and cultured in T cell media.
2. Centrifugation at 300 x g for 4 min and discard supernatant.
3. Add 2 mL of FSS to wash the cells, centrifuge at 300 x g for 4 min, and discard supernatant.
4. Stain the cells with appropriate antibody to indicate CAR expression at 4 °C for 30 min.
   NOTE: For example, if an IL13Rα2-targeted CAR is used, then stain with anti-IL13 antibody.
5. Wash twice with 2 mL of FSS and analyze CAR expression using a flow cytometer.
6. Determine the CAR% on T cells using the gating strategy shown in Figure 1B.
7. At the day of co-culture, harvest all CAR T cells by centrifugation at 300 x g for 4 min, discard supernatant, and resuspend cells in 2 mL of co-culture media.
8. Determine cell concentration using a cell viability counter.
   NOTE: Cell viability must be >70%.

4. Set up tumor — T Cell Co-culture

1. Dilute tumor cells to a concentration of 0.16 million/mL with co-culture media.
2. Based on CAR%, dilute CAR T cells to a concentration of 0.04 million CAR⁺ cells/mL with co-culture media.
   NOTE: For example, if the CAR is 50% and T cell concentration is 0.4 million/mL, then make a 1:5 dilution to get the final concentration of 0.04 million CAR⁺ cells/mL.
3. Pipette 100 µL of diluted tumor cells into each well of a 96-well flat-bottom tissue culture plate.
4. Pipette 100 µL of diluted CAR T cells into each well that contains tumor cells and mix well.
   NOTE: Each tumor-T cell co-culture will be analyzed at 4 time points. 4–6 replicates might be required at every time point based on different analysis, but <3 replicates per time point is not recommended; see Table 1 for a representative platemap.
5. Maintain the plate in a 37 °C, 5% CO₂ incubator.

5. Tumor Cell Rechallenge

NOTE: Rechallenge takes place at 2, 4 and 6 days post the initial co-culture setup (Figure 1A).

1. Harvest and dissociate GBM TSs as described above (steps 2.1–2.6).
2. Resuspend GBM cells at a concentration of 0.64 million/mL.
3. Determine the co-culture wells that need rechallenge (see Table 1) and carefully remove 50 µL media from the top of each well.
4. Add 50 µL of GBM cell suspension into each well and mix well, then put the plate back into a 37 °C, 5% CO₂ incubator.

6. Harvest Samples and Flow Cytometric Analysis

NOTE: Samples will be harvested at 1, 3, 5 and 7 days post the initial co-culture setup, with 1, 2, 3 and 4 rounds of tumor challenge, respectively.

1. Pre-warm 0.05% trypsin-EDTA solution in 37 °C waterbath.
2. Determine the wells that need harvesting and transfer the media into a new round-bottom 96-well plate.
3. Pipette 50 µL of trypsin-EDTA into the wells to digest remaining tumor cells at 37 °C for 5 min.
4. Under a microscope, confirm the cells have detached from the bottom.
5. Pipette around the well bottom to resuspend detached cells, then transfer trypsin-EDTA containing detached cells to the corresponding wells of the round-bottom 96-well plate.
6. Centrifuge the round-bottom 96-well plate at 300 x g, 4 °C for 4 min, then discard supernatant.
7. Add 200 µL/well of FSS to wash the cells, centrifuge at 300 x g, 4 °C for 4 min, then discard supernatant.
8. Resuspend cells in 100 µL/well FSS containing antibodies (see Table of Materials), and stain cells at 4 °C for 30 min.
9. Add 100 µL/well FSS to cells, centrifuge at 300 x g, 4 °C for 4 min, then discard supernatant.
10. Add 200 µL/well of FSS to wash the cells, centrifuge at 300 x g, 4 °C for 4 min, then discard supernatant.
11. Resuspend cells with 100–200 µL/well of FSS with 500 ng/mL DAPI, then analyze samples by flow cytometry.

Table 1: Representative plate map of rechallenge setup.