Ex Vivo T Cell Expansion Protocol

Thawed PBMCs were resuspended in TCGM (T cell growth media) composed of X-VIVO 15 (Lonza) supplemented with 10mM HEPS and 2mM Glutamax (both from ThermoFisher Scientific), 5% human AB serum (Valley Biomedical) and supplemented with 250 IU/mL recombinant human IL-2 (catalog 78220.3, Stemcell) before activation with 50 ng/mL anti-CD3 (clone OKT3) and anti-CD28 (clone 15E8) antibodies (Miltenyi Biotec). Lentivirus supernatants were added to PBMC cultures 24 hours later (multiplicity of infection = 10). Seventy-two hours after activation, transduced PBMCs were collected, washed, and resuspended in complete TCGM with human IL-2 at 0.5 × 10⁶ cells/mL and transferred to gas-permeable culture vessels (G-Rex, Wilson Wolf). PBMC cultures were expanded in vitro at cell density of 0.5 × 10⁶ to 2 × 10⁶ cells/mL maintained by the addition of fresh medium every 2–3 days for a total of 10–11 days until cryopreservation. Clinical T cell product manufacture was completed essentially as described previously (57 (link)), except T cell cultures were initiated with a 1:1 ratio of CD4⁺ and CD8⁺ T cells.