Ultrapure agarose hydrogel

Master 3D Petri dish 35-well arrays (Microtissues Inc., Providence, RI, USA) were used to create micro-wells made up of 2% UltraPureTM agarose hydrogel (Invitrogen, Carlsbad, CA, USA). Micro-wells were seeded with 10,000 cells/well. Cells used in this assay were PANC-1, BxPC-3, AsPC-1, HUVECs, and patient-derived pancreatic adenocarcinoma cancer fibroblast cells. After seeding, 1 mL of the appropriate medium was added, and templates were incubated at 37 °C with 5% CO₂ for 24 h up to 10 days. For hybrid spheroid experiments, pancreatic cancer cells were co-cultured with HUVECs and patient-derived pancreatic cancer fibroblast cells in the ratios: 3:1:1, respectively. Spheroids were formed using the 3D Petri dish templates as previously detailed. Since HUVECs are more sensitive to their environment compared with the other cells used in these experiments, and require an enriched medium for their growth, we used their optimized medium, as previously mentioned, for the co-cultured experiments. Light microscopy images were taken using Nikon Eclipse TS100 light microscope, x4 lens.

Spheroids were prepared as detailed in our previous work (Steinberg et al. 2020 (link)). Master 3D Petri dish 35-well arrays (Microtissues Inc., Providence, RI, USA) were used to create micro-wells made of 2% UltraPureTM agarose hydrogel (Invitrogen, Carlsbad, CA, USA). Micro-wells were seeded with 5,000 A375 or WM-266-4 cells per well. A375 and WM-266-4 cells were purchased from ATCC. The cells were incubated at 37 °C and 5% CO₂ for 72 h allowing for the growth of tumor spheroids.