X vivo 15 medium

According to the manufacturer’s instructions, we isolated CD3+ T cells from fresh cord blood by CD3 positive selection microbeads (Miltenyi Biotech, Germany). For activating the T cells, we resuspended the isolated CD3+ T cells (1 × 10⁶ cells/ml) in X-VIVO 15 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% human AB serum (Thermo Fisher Scientific) and 200 U/mL recombinant human IL-2 (PeproTech, USA) at 37°C in 5% CO₂. Sterile, non-tissue-culture-treated 24-well plates were coated with Retronectin (Thermo Fisher Scientific) at 6 µg/cm² and left to stand overnight at 37°C in 5% CO₂. Next, the lentivirus supernatant was transferred to plates, and then T cells activated using recombinant human interleukin-2 (250 U/mL) were added, followed by incubation at 37°C for 24h after centrifugation. The medium was changed 24h later and every other day afterwards.

K562 cells were cultured in RPMI medium + 10% heat-inactivated fetal bovine serum [HI FBS (Gibco/ThermoFisher; Waltham, MA)] + 1% penicillin, streptomycin, glutamine [PSQ (Gemini Bio-Products; Sacramento, CA)], and were kept at a density between 1 × 10⁵ and 1 × 10⁶ cells per ml. Healthy human CD34⁺ cells from mPB (peripheral blood stem cells, PBSCs) were thawed in pre-warmed X-Vivo 15 medium (Lonza; Basel, Switzerland) with 1% PSQ, pelleted at 500×g for 5 min, and resuspended at 5 × 10⁵ cells/mL in pre-warmed X-Vivo 15 medium with PSQ and SFT cytokines [50 ng/mL stem cell factor (SCF), 50 ng/mL fms-related tyrosine kinase 3 ligand (Flt3-L), and 50 ng/mL thrombopoietin (TPO)] (Peprotech; Rocky Hill, NJ). Cells were pre-stimulated at 37°C and 5% CO₂ incubator for 48 h.