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.