For early stage organoids that were cultured less than 1 month, dissociation was performed by using Accutase. Organoids were washed once with Hank's Balanced Salt Solution, then incubated with Accutase in 37°C water bath for 20 min. Accuta se was then removed and organoids were washed 3 times with HBSS. Gentle trituration was performed to obtain single cell suspension. For dissociation of older organoids, hMGEOs or hCOs were dissociated using the papain dissociation system according to the instructions from the manufacturer. All the solutions used were oxygenated for 5 min with 95% O2:5% CO2 to improve cell viability. Briefly, organoids were washed once with HBSS, then quickly dissected into small pieces in oxygenated papain solution. After dissection, the tissue suspension was re-equilibrated with 95% O2:5% CO2, and incubated in 37°C water bath for 60 min. The suspension was gently shaken every 20 min during the incubation period. Then gentle trituration was performed to obtain single cell suspension and papain was inactivated with albumin-ovomucoid inhibitor solution.
