Cancer cell culture

Human cancer cell lines CAOV3 and HCT-116 were cultured in DMEM (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). All human cell cultures were maintained at 5% CO2 in a humidified 37°C incubator. 

Preparation and expansion of CAR-T cells
PBMCs were isolated from buffy coat samples from healthy donors (National University of Singapore, NUS-IRB B-14-133E) or blood samples from patients with ovarian cancer (Cancer Hospital of the University of Chinese Academy of Sciences, IRB-2020-15) with Ficoll- Paque-based density gradient centrifugation. For optimization
of T cell activation, PBMCs were activated with soluble OKT3 anti- CD3 monoclonal antibody (eBioscience, San Diego, CA, USA) at either 100 ng/mL or 500 ng/mL and cultured in AIM-V (Invitrogen, Carlsbad, CA, USA) supplemented with 5% AB serum (Gemini Bio, West Sacramento, CA, USA) and 300 IU/mL IL-2 (Miltenyi Biotec, Germany) for 2 or 3 days. OKT3-activated PBMCs were then harvested and washed thrice with OptiMEM (Invitrogen) before resuspension in P3 Primary Cell Nucleofector Solution (Lonza) at a cell density of 1 x 10^8 cells/mL. For each electroporation reaction with 100 µL of cell suspension, 5 µg of piggyBac (PB) transposase plasmid and one of the following transposon donor plasmids were added at the indicated amounts: 10 µg of aCD22bp, 10 µg of NKG2D, or 30 µg of NKG2D-CD20.37 Cells and DNA mixtures were transferred to a 100-µL single Nucleocuvette (Lonza) and electroporated with the EO-115 program of the 4D-Nucleofector system (Lonza).