Intesticult basal medium

Following the collection of gastric tissue, tissue was cut into small pieces and transferred to a 50 mL Falcon tube containing 20 mL of room temperature Gentle Cell Dissociation Reagent (Stem Cell technologies). The samples were then incubated at room temperature for 20 min on an orbital roller. Glands were released from the underlying tissue by shaking the tube vigorously for 20 seconds and supernatant was transferred to a new Falcon tube and centrifuged for 5 min at 1500 rpm (500 g) (4˚C). The supernatant was tipped off and the pellet was resuspended in 1 mL Advanced DMEM-F12 with Penicillin/Streptomycin (1/100) and passed through a 70 µm cell strainer. The number of glands in 10 µl was counted with a microscope, to determine the volume needed to have 100 glands per 50µl of Matrigel. Glands were centrifuged for 5 min at 1500 rpm (500 g) (4 °C) and supernatant was discarded. Glands were then resuspended in Matrigel, and 50 µl was pipetted into each well of a pre-warmed 24-well plate to create domes. 500 µl of IntestiCult™ Basal Medium (Stem Cell technologies) was added to each well, comprised of 90 ml IntestiCult™ Basal Medium, supplemented with 5 ml of the IntestiCult Supplement 1, 5 ml of Supplement 2 and Penicillin/Streptomycin (1/100). Organoids were grown at 37 °C, 10% CO₂ in a humidified incubator, changing the media every 3 days.