Macs gmp t cell transact cd3 cd28 beads

To prepare T cells, healthy donor blood samples were obtained under appropriate safety and ethics approvals by the Ottawa Hospital Research Institute (Ottawa, Canada). Whole blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and PBMCs were isolated by Ficoll-Paque^™ density gradient centrifugation, centrifuging for 20 min at 700 × g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 × g. PBMCs were resuspended and counted by mixing 1:1 with Cellometer ViaStain^™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, Massachusetts, USA). T cells from were then activated with Miltenyi MACS GMP T cell TransAct^™ CD3/CD28 beads and seeded 1×10⁶ T cells/ml in serum-free StemCell Immunocult^™-XF media (Cat#10981, StemCell Technologies, Vancouver, Canada) with 20U/ml clinical grade human IL-2 (Novartis). T cells were then polyclonally expanded for 7 to 10 days in culture before cryopreservation of aliquots using complete media + 10%DMSO.

Heparinized whole blood was collected from healthy donors under informed consent by venipuncture and transported at room temperature from Ottawa Hospital Research Institute. Blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and PBMCs were isolated by Ficoll-Paque™ density gradient centrifugation. Briefly, samples layered on Ficoll-Paque™ gradient were centrifuged for 20 min at 700 × g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 × g. PBMCs were resuspended and counted by mixed 1:1 with Cellometer ViaStain™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, MA, USA). T cells from were then activated with Miltenyi MACS GMP T cell TransAct™ CD3/CD28 beads and seeded 1x10⁶ T cells/mL in serum-free StemCell Immunocult™-XF media (StemCell Technologies, Vancouver, Canada) with clinical grade 20 U/mL human IL-2 (Novartis, Basel, Switzerland).