Detailed description of ductal and acini organoid induction from pancreatic progenitors is previously described (Huang L et al., 2021 (link)). Cell culture plates or 8-well falcon chamber slides were coated with undiluted Matrigel and incubate at 37°C to solidify. Transduced or uninfected pancreatic progenitors were seeded as single cells on Matrigel-coated plates in appropriate differentiation media (described in Huang et al., 2021 (link)) containing 5% Matrigel. Media change was performed every 4 days.
For long-term culture of GNASR201C-expressing organoids, Day16 ductal organoids were split in a 1:3 ratio using 1mg/ml Collagenase/Dispase (Sigma) in DMEM-HG (Gibco) for 30min-1hr, followed by mechanical dissociation of organoids using a p1000 pipette. After centrifugation (1000rpm, 5min), dissociated organoids were embedded in 100% Matrigel and seeded as 80μl domes in 24-well cell culture plates. The domes were allowed to solidify for 30min at 37°C. Stage 4 ductal differentiation media containing doxycycline (1μg/ml) was used to culture GNASR201C-expressing organoids. Culture media was replaced every four days and organoids were split in a 1:3 ratio every 12–16days.
For long-term culture of GNASR201C-expressing organoids, Day16 ductal organoids were split in a 1:3 ratio using 1mg/ml Collagenase/Dispase (Sigma) in DMEM-HG (Gibco) for 30min-1hr, followed by mechanical dissociation of organoids using a p1000 pipette. After centrifugation (1000rpm, 5min), dissociated organoids were embedded in 100% Matrigel and seeded as 80μl domes in 24-well cell culture plates. The domes were allowed to solidify for 30min at 37°C. Stage 4 ductal differentiation media containing doxycycline (1μg/ml) was used to culture GNASR201C-expressing organoids. Culture media was replaced every four days and organoids were split in a 1:3 ratio every 12–16days.
