Cd8 positive magnetic bead separation

Fresh umbilical CB was collected after informed consent was obtained according to the Declaration of Helsinki. CB was processed to isolate CD8⁺ T cells using Ficoll (GE Healthcare Bio-Sciences AB) separation and CD8-positive magnetic bead separation according to the manufacturer’s protocol (Miltenyi Biotec). CD8⁺ T cells were subsequently cultured in cytotoxic T lymphocyte (CTL) media (consisting of RPMI (Fisher Scientific) supplemented with 10% human serum, 1% penicillin/streptomycin (P/S), 50 U of IL (Interleukin)-2/mL (Proleukin, UMCU Pharmacy), 5 ng/mL IL-7, and 5 ng/mL IL-15 (Miltenyi Biotec)) and activated for 3 days with anti CD3/CD28 Dynabeads (GIBCO, Thermo Fisher Scientific) at a 1:3 ratio (beads:T cells).

Fresh CB or PB was collected after informed consent was obtained according to the Declaration of Helsinki. The collection protocol was approved by the Ethical Committee of the University Medical Center (UMC) Utrecht. CB or PB was processed to isolate CD8⁺ T cells using Ficoll (GE Healthcare Bio-Sciences AB) separation and CD8-positive magnetic bead separation according to the manufacturer’s protocol (Miltenyi Biotec). CD8⁺ T cells were subsequently cultured in RPMI (Fisher Scientific), supplemented with 10% human serum; 1% penicillin/streptomycin (P/S) (cytotoxic T lymphocyte media [CTL media]), with aCD3/CD28 Dynabeads (Gibco; Thermo Fisher Scientific) in a 1:3 ratio (beads:T cells); 50 U of IL-2/mL (UMC-Pharmacy); 5 ng/mL IL-7; and 5 ng/mL IL-15 (Miltenyi Biotec). CD8⁺ T cells were kept in culture for 3 days, resulting in an average 4.5-fold expansion rate.