Buffy coats from consenting healthy anonymous donors were obtained from the German Red Cross Dortmund as registered and approved by the Ethics Committee of the German Red Cross. All study-related procedures have been performed according to the Declaration of Helsinki and to the relevant ethical guidelines.
CAR T cells were generated according to previously applied protocols 46 (link). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation and subsequent T cells enrichment was performed using the human pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Isolated T cells were cultivated in TexMACSTM Medium (Miltenyi Biotec) supplemented with 200 IU/ml of recombinant human IL-2 IS, research grade (Miltenyi Biotec) and activated with TransActTM, human (Miltenyi Biotec). Twenty-four hours after activation, lentiviral vector was added for transduction. T Cell TransActTM and excess viral vector were removed 3 days post activation and replaced with fresh TexMACSTM Medium, supplemented with 200 IU/mL IL-2, for further cultivation of T cells. Cell splitting and feeding occurred in a 2-3 days interval. After 12 days of expansion, T cells were subjected to experimental studies.
CAR T cells were generated according to previously applied protocols 46 (link). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation and subsequent T cells enrichment was performed using the human pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Isolated T cells were cultivated in TexMACSTM Medium (Miltenyi Biotec) supplemented with 200 IU/ml of recombinant human IL-2 IS, research grade (Miltenyi Biotec) and activated with TransActTM, human (Miltenyi Biotec). Twenty-four hours after activation, lentiviral vector was added for transduction. T Cell TransActTM and excess viral vector were removed 3 days post activation and replaced with fresh TexMACSTM Medium, supplemented with 200 IU/mL IL-2, for further cultivation of T cells. Cell splitting and feeding occurred in a 2-3 days interval. After 12 days of expansion, T cells were subjected to experimental studies.
Free full text: Click here
