2.7. Tissue Dissociation and Organoid Culture

Patient-derived organoids were isolated and cultured using minor modifications of a protocol previously published by Lee et al. [14]. Tumour tissues from patients were washed in PBS containing penicillin/streptomycin. Tumour tissues were minced with scissors and incubated in 10 mL of the organoid culture medium (hepatocyte medium with 10 ng/mL EGF, 5% charcoal-stripped FBS, 10 mM Y-27632, 100 mg/mL Primocin, and 1× Glutamax) supplemented with 1 mL collagenase/hyaluronidase (STEMCELL Technologies) at 37 °C for 15 min. Dissociated tissues were spun down at 300 g for 5 min, resuspended in 10 mL of PBS, and spun down again. The tissues were resuspended in 5 mL of TrypLE Express (Invitrogen, Life Technologies, Carlsbad, CA, USA) and incubated at room temperature for 3 min. Dissociated tissues were spun down at 300 g for 5 min, resuspended in 10 mL of HBSS supplemented with 5% charcoal-stripped FBS, 10 mM Y-27632 and 100 mg/mL Primocin, and passed through a 100 μm cell strainer. Dissociated cells (1 × 10⁶ cells/well) were spun down at 300 g for 5 min, resuspended in 60% Matrigel/organoid culture medium, plated in a 250 μL drop in the middle of one well of a pre-coated 6-well plate with 60% Matrigel, and solidified at 37 °C for 30 min. After solid drops formed, 1.5 mL of the organoid culture media was added to the well.