Rocki

After two passages on Matrigel, the RH6 cells were transferred to 125 mL spinner flask
(Cellspin; Integra Biosciences AG, Switzerland) at a 40rpm agitation rate. For large-scale
expansion, a 100-ml working volume was used as previously described (29 (link)). Briefly,
undifferentiated RH6 cells were cultured with the optimal starting concentration of
2−3×10⁵ cells/mL at the hESC media, which was conditioned by MEFs, fresh 10
mM Rhoassociated kinase inhibitor (ROCKi; Sigma, Netherlands) and 100 ng/mL bFGF. The
spinner flask was placed on a magnetic stir plate in an incubator at 37˚C and 5%
CO₂ without changing media during the first two days. RH6 cells were expanded
in a 3D-dynamic suspension culture after 4 days.

In this experimental study, hESCs (RH5 line) were
cultured and expanded as spheroids according to a
previously described protocol (22 (link)). Briefly, 2×10⁵
viable cells/ml were cultured in hESC medium that
consisted of Dulbecco’s Modified Eagle Medium/
Ham’s F-12 (DMEM/F12, Gibco, USA) supplemented
with 20% knockout serum replacement (KOSR, Gibco,
USA), 1% insulin-transferrin-selenite (Gibco, USA),
1% nonessential amino-acids (NEAA, Gibco, USA),
1% penicillin/streptomycin (Gibco, USA), 0.1 mM
ß-mercaptoethanol (Sigma-Aldrich, USA), and 100 ng/
ml basic fibroblast growth factor (bFGF, Royan Biotech,
Iran) in non-adhesive bacterial plates. The medium was
renewed every 2 days. When spheroids reached 200-250
µm, they were dissociated into single cells with Accutase
solution (Sigma-Aldrich, USA), and replated on new
bacterial plates at a 1:3 ratio. Cells were treated with 10
µM of ROCK inhibitor (ROCKi, Sigma-Aldrich, USA)
for the first 2 days.

Pancreatic organoids infection was performed as described in^{21 (link)}. Viruses were produced in HEK293T cells and used at multiplicity of infection of 10 for the transgene or 20 for the transctivator Tet-On® 3G. Organoids were dissociated into small clusters, incubated with culture media containing the viruses, 8 μg/ml polybrene (Sigma), 10 μM ROCKi (Sigma) and span for 1 h at 300 × G at room temperature. After the spin, infected organoids were incubated in a cell culture incubator at 37 °C for 5–6 hours before plating in matrigel with fresh media supplemented with ROCKi.