Cls3471

1 × 10⁶ cells were seeded in ultra-low attachment plates (Corning Costar, CLS3471) for 48h. For treatments, cells were collected, centrifuged at 50 x g for 5mins and washed in PBS. Cells were then resuspended in 2ml of treatment medium and transferred back into ultra-low attachment plates for indicated treatment times.

The patient-derived organoid culture protocol was adopted from literature^{74 (link)} with some modifications. Briefly, organoids were dissociated into smaller clumps of cells and split into an ultra-low attachment 6-well plate (Costar, CLS3471). Organoid cells were then transduced with viruses and incubated at 37 °C overnight. The next day, the virus-transduced cells were collected and resuspended in the 1:1 mixture of cold cultrex growth factor reduced BME type 2 (R&D system, 3533-010-02) and organoid medium^{74 (link)}, and left to solidify at room temperature for 30 min. Upon completed gelation, an organoid medium containing 1 µg/ml puromycin or blasticidin was added to each well to select stable transduced cells. The stable transduced organoid cells were then dissociated and re-seeded into 24-well suspension plates at the same ratio for each group. The number of organoids was counted on days 3–5 under the microscope. Generation of PDO was done in compliance with all relevant ethical regulations, with informed consent from the patient, and approved by the National Healthcare Group DSRB (reference number: 2015/00357).