Tumor-Infiltrating Lymphocyte Isolation and Expansion

The procedure was adapted from Kondo et al. (2011) [11 (link)] and modified as follows. Tumor specimens were washed in HBSS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), minced with forceps, and digested with LiberaseTM DH [12 (link)] for 2 h at 37 °C. Digested tissue was centrifuged (300× g, 5 min), washed with HBSS and filtered through a stainless 500 μm steel mesh (VWR). The flow-through was again filtered through a 40 μm cell strainer (Corning, Corning, NY, USA). The filtrate containing the TIL fraction was resuspended in Advanced RPMI 1640 (Gibco) supplemented with 2 mM Glutamine (Gibco), 1% MEM Vitamins (Gibco), 5% human serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL primocin (Invivogen, San Diego, CA, USA). IL-2 (100 U/mL), IL-7 (10 U/mL) and IL-15 (23.8 U/mL) (Peprotech, East Windsor, NJ, USA) were freshly added to culture media. For expansion, CD3/CD28 dynabeads were added (Milteny Biotech, Auburn, CA, USA). PDM, held back by cell strainer, were washed in HBSS and cultured in suspension in StemPro^® hESC SFM (Gibco) supplemented with 8 ng/mL FGF-basic (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1.8% BSA (Gibco) and 100 μg/mL primocin (Invivogen) within cell-repellent culture dish (60 × 15 mm) (Corning).