Panc-1 Tumor Spheroid Generation

Spheroids were obtained as previously described (Zanoni et al., 2016 (link)). Briefly, a rotatory cell culture system RCCS (Synthecon Inc., Houston, TX, United States) was used. The rotary systems were placed inside a humidified 37°C, 5% CO₂ incubator and all procedures were performed in sterile conditions. Single cell suspensions of about 1 × 10⁶ cells/ml of Panc-1 were placed in the 50 mL rotating chamber at an initial speed of 12 rpm. Speed was increased as cells formed aggregates to avoid sedimentation. The culture medium was changed every 4 days and tumor spheroids with an equivalent diameter ranging from about 500–1300 μm were obtained in around 15 days. After formation, spheroids were transferred into a 96-well low-attachment culture plates (Corning Inc., Corning, NY, United States; one spheroid/well), containing 100 μL of fresh culture medium per well.