Easysep human t cell isolation kit

Peripheral blood leukapheresis products were obtained from Department of Haematology, Singapore General Hospital (Central Institutional Review Board [CIRB] reference: 2019/2600). Human T cells from leukapheresis samples were isolated using EasySep^TM Human T Cell Isolation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada). Viable human T cells were seeded in fresh AIM V supplemented with 2% AB human serum and interleukin-2 (IL-2) (100 IU/mL). To activate T cells, 25 μL of ImmunoCult^TM Human CD3/CD28 T cell activator was added to 1 mL of 1 million of cell suspension and incubated at 37°C with 5% CO₂. After 3 d of activation, a viable cell count was performed and the viable cell density was adjusted every 2–3 d by adding fresh complete AIM V + 2% AB human serum + IL-2 (100 IU/mL) to the cell suspension. The cells were incubated at 37°C with 5% CO₂ until the desired cell number is obtained or for up to 12 d.

CD8⁺ and CD4⁺ T cells were isolated from healthy donor leukaphereses using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies). Isolated CD8⁺ or CD4⁺ T cells were activated using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) and cultured in RPMI 1640 with 10% human serum, 3.51 mM L-glutamine, 0.05 mM β-mercaptoethanol, 100 U/mL penicillin-streptomycin, and 50 IU/mL IL-2 (PeproTech). After 24 hours, activated T cells were spinoculated at 800g and 32°C for 90 minutes with a lentivirus encoding a CAR comprising an FMC63-derived CD19-specific single-chain variable fragment (scFv), 4-1BB costimulatory domain, and CD3ζ signaling domain. A truncated cell-surface human epidermal growth factor receptor (EGFRt) was separated from the CAR using a ribosomal skip sequence to enable the detection of transgene expressing cells by flow cytometry. Dynabeads were removed on day 5. Transduced EGFRt⁺CD8⁺ and EGFRt⁺CD4⁺ T cells were flow-sorted (BD FACSAria or Sony MA900) on day 7 and expanded by coculture with irradiated allogeneic CD19⁺ lymphoblastoid cell line for 8 days. Media were changed every 2 days. CAR T cells were used for further experiments on days 14 to 16.