Dynabeads untouched human t cells isolation kit

Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by gradient density centrifugation on a Ficoll-Paque (GE Healthcare) according to standard protocol. Peripheral blood of patients and healthy donors was obtained from Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology. All healthy donors provided informed consent. Dynabeads Untouched Human T-cells Isolation Kit (Invitrogen) was used for isolation of T cells from PBMCs. T cells were activated with CD3/CD28 Dynabeads (Thermo Scientific) at a 1:1 ratio in a media containing 40 IU/ml recombinant IL-2 (Pan Biotech) for 24 h. Activated T cells were transduced using lentiviral viral sups (CAR19 or CAR19-T2A-VHH-PEBL) according to protocol presented above for Jurkat cells. Transduced cells were grown in flasks at a density of 1.0×10⁶/ml, culture medium was replaced every 2 days.

The lentiviral particles were prepared by PEI co-transfection of HEK293T cells with the corresponding lentiviral CAR plasmids and the packaging plasmids (p-VSV-G, pGAG and pREV). The lentiviral supernatants were collected 48 h after transfection. Peripheral blood of patients and healthy donors was obtained from Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology. Dynabeads Untouched Human T Cells Isolation Kit (Invitrogen, USA) was utilized for isolation of T cells from human PBMCs. Human T cells were activated with CD3/CD28 Dynabeads (Thermo Scientific, USA) at a 1:1 ratio (Life Technologies, USA) in a complete RPMI containing 40 IU/mL recombinant IL-2 (Pan Biotech, USA) for 24 h. Activated T cells were re-suspended at concentration of 0.5 × 10⁶/mL in lentiviral supernatant of CD19-CAR 1 mL of fresh RPMI media supplemented with 30 IU/ml IL-2 was added, and cells were plated to 6-well plates. Plates were centrifuged at 1200g for 90 min at 32 °C and incubated for 6 h at 37 °C. Then culture medium was changed every 2 days and cells were grown in flasks at a density of 2.0 × 10⁶/mL.