2.7 T Cell Expansion
CD4+ Naïve cells were obtained from Astarte Biologics (Astarte Biologics, Washington, USA) and were cultured in X-VIVO™ serum free T-Cell medium (Lonza, New Jersey, USA). This medium was additionally supplemented with recombinant human interleukin 2 (IL-2), recombinant human interleukin 7 (IL-7), and recombinant human interleukin 15 (IL-15) (Miltenyi Biotech GmbH, Germany) at various concentrations on Day 3. In the ‘000’ condition, medium was supplemented with the following interleukin concentrations: IL-2 (1 ng/mL), IL-7 (6 ng/mL), IL-15 (6 ng/mL). In the ‘110’ condition, medium was supplemented with the following interleukin concentrations: IL-2 (10 ng/mL), IL-7 (36 ng/mL), IL-15 (6 ng/mL). T cells were combined with anti-CD3/anti-CD28 DynaBeads® (Gibco, Waltham, MA) in bead:T cell ratio of 1:1. Cells were then seeded at 166,667 cells per mL in the described surface-attached microplate with 300 µL culture medium. The day of seeding is referred to as Day 0 of the expansion. Cell samples were obtained in the process daily starting on Day 3 for enumeration.
During expansion, mixed condition was subject to agitation via surface-attached microposts which were agitated via magnetic motor. The motor was operated at 4000 rpm 5 minutes on 55 minutes off cycle continuously throughout the duration of expansion.