4.2. Tumoroid Culture

Tumoroids were formed in the 3D culture systems using NanoCulture Plate (NCP) (Medical & Biological Laboratories, Nagoya, Japan) or ultra-low attachment (ULA) culture plates/dishes (Greiner, Kremsmunster, Austria) within mTeSR1 stem-cell medium (Stemcell Technologies, Vancouver, BC, Canada) or the above-mentioned serum-containing medium as described previously [34,35,36,42].

For quantification of tumoroids size and number, cells were seeded in a 96 well NCP for 14 days at a concentration of 5.0 × 10³ cells in 200 μL mTeSR1 or RPMI-1640 media with 10% FBS. Tumoroid maturation was monitored every day and photographed using the Floid cell imaging station (Thermo Fisher, Waltham, MA, USA) from day 1 until day 7 and a BZ-X microscope (Keyence, Osaka, Japan) starting from day 10 until the end of the experiment day 14. The tumoroid size was measured using Image J software (NIH, Bethesda, MD, USA).