CAR-T Cell Manufacturing Protocol

CAR-T cells were manufactured as described in our previous study (23 (link)). The detailed methods were as follows: peripheral blood mononuclear cells (PBMCs) were collected and isolated by Ficoll density gradient centrifugation. CD3⁺ T cells were selected by CD3 microbeads (Miltenyi Biotec, Cambridge, MA, USA), stimulated by anti-CD3/anti-CD28 mAb-coated Human T-Expander beads (Cat.no. 11141D; Thermo Fisher Scientific, Waltham, MA, USA) and cultured in T-cell medium X-Vivo 15 (Lonza Group, Ltd., Basel, Switzerland) supplemented with 250 IU/ml interleukin-2 (IL-2; Proleukin; Novartis International AG, Basel, Switzerland). All the CD3⁺ T cells (3 × 10⁶) were transduced with a lentiviral vector encoding humanized CD19 CAR constructs (10 μg, lenti-CD19-2rd-CAR; Shanghai Genbase Biotechnology Co., Ltd., Shanghai, China) and cultured in media containing recombinant human IL-2 (250 IU/ml). On the 12th day of cultivation, transduction efficiencies of anti-CD19-CAR were analyzed by flow cytometry (FCM) (BD Biosciences, San Jose, CA, USA).