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Isolation, activation, transduction, and expansion of MET-CAR-T

CC Cristina Chiriaco
CD Chiara Donini
MC Marco Cortese
SU Stefano Ughetto
CM Chiara Modica
IM Ilaria Martinelli
AP Alessia Proment
LV Letizia Vitali
LF Lara Fontani
MC Monica Casucci
PC Paolo Maria Comoglio
SG Silvia Giordano
DS Dario Sangiolo
VL Valeria Leuci
EV Elisa Vigna
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T cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donors or of patients with gastric carcinoma. PBMC were isolated by density gradient centrifugation (Lymphosep, Aurogene) and then were activated by anti-Biotin MACSi Bead Particles loaded with anti-CD2, anti-CD3, and anti-CD28 antibodies (Miltenyi Biotec) for 24 hrs. PBMC were maintained in culture medium + human recombinant IL-2 (100 U/mL, Miltenyi Biotech) for a further 24 hrs and then transduced with MET-CAR-LVs (MOI 10) for 8 hrs in the presence of polybrene (8 μg/mL). Not-transduced T cells (NTD) were used as a paired control. CAR-T or NTD-T cells were expanded for a maximum of 3 weeks in the presence of IL-2 (100 U/mL). Transduction efficiency (evaluated by eGFP expression) ranged from 30 to 70% of the total population.

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