96 well low attachment culture plates

A rotatory cell culture system (RCCS) (Synthecon Inc., Houston, TX, USA) was used35 (link). The rotator bases were placed inside a humidified 37 °C, 5% CO2 incubator and connected to power supplies set up externally to the incubator. All procedures were performed in sterile conditions under a laminar flow hood. Single cell suspensions of about 1 × 10⁶ cells/ml of A549 were placed in the 50-ml rotating chamber at an initial speed of 12 rpm. As the majority of cells formed aggregates and these aggregates gradually enlarged, speed was increased over time to avoid aggregate sedimentation within the culture vessels which could hinder complete spheroid formation. The culture medium was changed every 4 days and tumor spheroids with an equivalent diameter ranging from about 500–1300 μm (depending on the cell line used) were obtained in around 15 days. After the formation of the spheroids, the operator, working under the sterile laminar flow hood, transferred spheroids to 96-well low-attachment culture plates (Corning Inc., Corning, NY, USA) (one spheroid/well), each well previously filled with 100 μl of fresh culture medium.

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.