Optmizer t cell expansion supplement

Informed consent was obtained prior to collection of peripheral blood from healthy adult donors. PBMC were isolated immediately via density gradient centrifugation using Lymphoprep™ (Axis Shield, Norway) following manufacturer’s instructions. PBMCs were resuspended to 1 × 10⁶/mL in CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies, Australia) supplemented with OpTmizer™ T cell Expansion Supplement (1:38 dilution) (Life Technologies, Australia), 10% heat-inactivated FBS (HI-FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mmol L-glutamine (Life Technologies, Australia), 25 mM HEPES, 0.1% β-mercaptoethanol (Sigma–Aldrich, USA), 100 IU/mL recombinant human interleukin 2 (rhIL-2) (BD Pharmingen, USA) and activated with 5 μM ZOL, and seeded into 6-well plates. Cell culture density was maintained at 1–2 × 10⁶ cells/mL and replenished with fresh medium containing 100 IU/mL rhIL-2 only (without ZOL) every 2–3 days. Following 7–8 days of culture cells were collected and enriched as described below.

Whole blood was collected from healthy donors through the Emory Children’s Clinical and Translational Discovery Core (IRB0010797) and peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque Plus (GE Healthcare Life Sciences) density centrifugation. PBMCs were cultured in OpTmizer media supplemented with OpTmizer T-cell expansion supplement (Life Technologies), 1% penicillin/streptomycin, and 2 mM L-glutamine. Cell counts were performed using a Cellometer (Nexelcom) and cells were resuspended in fresh media at 1.5×10⁶ cells/mL every 3 days. To selectively expand γδ T cells from PBMCs, 5 μM of Zoledronate and 500 IU/mL of IL-2 were added on day 0 and 3 of expansion. αβ T cells were depleted from the culture on day 6 of expansion using a GMP-compliant protocol (Miltenyi Biotec). Briefly, cells were washed in autoMACS Rinsing Solution containing 0.5% BSA (Miltenyi Biotec), incubated with Anti-TCRα/β-Biotin for 10 minutes at 4°C, washed in autoMACS Rinsing Solution and then filtered through a 0.4μM filter. Cells were then incubated with Anti-Biotin Microbeads (Miltenyi Biotec) for 15 minutes at 4°C, washed in autoMACS Rinsing Solution and passed through an LD Column (Miltenyi Biotec). Post-depletion, 1000 IU/mL of IL-2 was added on day 6 and 9 of expansion.