Isolation and Expansion of Melanoma TIL

TIL were obtained from melanoma lesions after patient's informed consent. Patients were HLA-typed, either based on a TIL or a PBMC sample. After mechanical tissue disruption, TIL were cultured for 15–40 days (to reach 5 × 10⁶ cells) in RPMI 1640 (Invitrogen) with 10% heat-inactivated human AB serum (Sigma), 1% Penicillin/Streptomycin (Invitrogen), fungizone® 1,25 μg/ml (Bristol-Myers Squibb) and IL-2 6000 IU/ml (Proleukin®, Novartis). Afterward, rapid expansion was performed for 14 days in the presence of allogenic feeders 1.3 × 10⁶/ml (40 Gy irradiated PBMC from two healthy donors), CD3 antibody 30 ng/ml (OKT-3, Thermofisher), and IL-2 6000 IU/ml in 50% RPMI and 50% AIM-V medium (Invitrogen), supplemented with 10% heat-inactivated human AB serum (25 (link)). TIL were frozen at 15–50 × 10⁶ cells/vial, using the same procedure as for the PBMC samples. Viability after thawing was >60% in all cases.