Fetal clone 3

Cystic organoids formed from PDAC93-GFP and solid organoids formed from PM12500-GFP pancreatic ductal adenocarcinoma (PDAC) cells derived from Pdx1-Cre; Kras+/LSL-G12D; Trp53+/LSL-R172H male mice (KPC) were kindly donated by Dr. Mariano Ponz and Dr. Silvestre Vicent. Authentication of cell lines was not required. The cell line used to generate the organoids was a primary line donated by a collaborator, generated from a transgenic mouse model of pancreatic cancer. It is not a commercial line, thus authentication is not possible. The organoid cell line was tested for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kit (Lonza). Cells were thawed from frozen stock in 50 ml Falcon tubes containing 10 ml of DMEM and centrifuged to remove any traces of the cryoprotectant DMSO. Then, the cells were incubated in T75 flasks (TC treated; Nunc EasyFlask, Thermo Scientific) containing 10 ml of DMEM supplemented with 10% FetalClone III (SH30109.02, Cytiva) at 37 °C and 5% CO2. Once cells reached 90% confluence, cell cultures were passed to a new T75 flask using 0.05% Trypsin-EDTA (25300-096, Gibco) followed by centrifugation and resuspension in 10 ml of fresh DMEM supplemented with serum and subsequent incubation at 37 °C and 5% CO2.

PDAC93-GFP and PM12500-GFP cells were grown in T75 flasks (TC treated; Nunc EasyFlask, Thermo Scientific) containing 10 ml of DMEM supplemented with 10% FetalClone III (SH30109.02, Cytiva). Once the cells reached 90% confluence, they were detached using 0.05% trypsin-EDTA (25300-096, Gibco), centrifuged, and resuspended in 1 $ml$ of fresh DMEM supplemented with serum. Then, the cell suspensions were embedded in 4 mg/ml growth factor reduced (GFR) Matrigel at a final concentration of 300,000 cells/ml. A total of 20 $\mu l$ of this cell-hydrogel mixture was added to individual 5 mm diameter wells of a homemade PDMS device followed by a 15 min incubation at 37 °C to complete the Matrigel gelation process. Finally, 800 $\mu l$ of organoid feeding media (a detailed description of the cell culture media used is available in Table S4) was added to the organoid culture device, and the 3D cell culture was incubated at 37 °C and 5% $C{O}_2$ as needed.