Figure 5. Timeline of Treg isolation and ex-vivo expansion.

The flowchart shows the procedure from the receipt of leukapheresis product, through CD4⁺ cells separation on CliniMACS^® device, Treg sorting (details of sorting strategy is described in Figure 4), Treg expansion, collection and preparation of Final Treg Product with indication of Quality Check Points during the process. During 48-hour sorting process, samples with sorted Tregs were centrifuged and collected, Tregs were re-suspended in Treg Culture Medium with IL-2 for short-term culture. After sorting, Tregs were pooled together, counted and distributed into 96-well U-bottom plates at concentration 2 × 10⁵ cells per well. Next, anti-CD3/CD28-coated beads were added and Tregs were expanded for 13 days. Treg culture was maintained on daily basis and Tregs were split with addition of fresh medium with IL-2. On day 7, Tregs from all wells were collected, sample of Tregs was retrieved for phenotype and viability analysis as Quality Check of expansion process. Then, Tregs were counted, re-distributed on plates and re-stimulated with fresh anti-CD3/CD28-coated beads. At day 13 of expansion, Tregs were collected, suspended in Final Treg Product Medium and anti-CD3/CD28 beads were removed on ClinExVivo^™ MPC^® magnet. After bead removal, sample of Treg cell suspension was taken for Final Treg Product Testing.