L ascorbic acid 2 phosphate

Cell suspensions after TIL isolation were cultured in TIL expansion medium with cytokines for 14 days. The TIL expansion medium consisted of MEM alpha (Invitrogen), 15% purified autologous serum or pooled heat-inactivated human AB serum (COSMO BIO), 2 mM GlutaMAX (Thermo Fisher Scientific), 1% insulin–transferrin–selenium (Thermo Fisher Scientific), 50 ng/ml l-ascorbic acid 2-phosphate (Nacalai), 0.5% 45 w/v% D (+)-glucose (Wako), 1% penicillin–streptomycin–amphotericin B suspension or 1% antibiotic–antimycotic mixed stock solution, and 50 ng/ml gentamicin sulfate. TIL were expanded in three cytokine conditions: IL-2 alone, IL-2 + IL-7 + IL-15 + IL-21, and IL-2 + IL-7 + IL-15 + IL-21 + IL-12 + IL-18. The final concentrations of the cytokines were 6000 U/ml IL-2 (Peprotech), 5 ng/ml IL-7 (Peprotech), 5 ng/ml IL-15 (Peprotech), 20 ng/ml IL-21 (Peprotech), 50 ng/ml IL-12 (Merck), and 50 ng/ml IL-18 (MBL). The cultures were split or scaled up to maintain less than 80% confluence or half of the culture medium was replaced every 2 to 3 days. Cells were counted in trypan blue and analyzed by flow cytometry after 14 days of expansion.