Establishment and culture of PDPCs

The spatially distinct multi-regions of the operable GBC tissue samples were collected from seven patients and PDPCs were established by following the standard procedures. In brief, fresh tissues were washed with sterile phosphate-buffered saline (PBS) and cut into 1 mm³ pieces. Then, small pieces of the tissue were placed into 10 cm² dishes and incubated with DMEM/F12 medium with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% non-essential amino acids, and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were trypsinized with 0.25% trypsin (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) when they reached about 30% confluence and were transferred into 24-well plates for subculture. Thereafter, all PDPCs were grown in a 37 °C incubator at 5% CO₂ with DMEM/F12 medium supplemented with 10% FBS. The mycoplasma testing has been done for all PDPCs established in this study. The passage of each PDPC used in the following experiment was different and the passage information for each PDPC in the assays ranged between 15 and 25.