Dmem 4.5 g l glucose

mPAC cells were grown in DMEM-4.5 g/L glucose (Sigma-Aldrich, St Louis, MO, USA) plus antibiotics supplemented with 10% fetal bovine serum. INS1E cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol. For adenoviral transduction experiments, cells were seeded onto 6-well plates and treated one day later with adenoviruses at a multiplicity of infection (moi) of 40 unless otherwise indicated for 2 h. Then, virus containing-media was replaced and cells were cultured for the indicated periods.
The coding sequence of the mouse Wnt9a cDNA was amplified from embryonic (E15.5) brain with the primers listed in Supplementary material and cloned into the adenoviral pAC.CMV shuttle vector. The recombinant adenovirus was constructed by homologous recombination in HEK293 cells. The adenovirus encoding human TCF7L253 (link) was kindly provided by Dr. C. Fillat (IDIBAPS, Barcelona, Spain). All other adenoviruses were previously described21 (link)65 (link).

mPAC cells were grown in DMEM-4.5 g/L glucose (Sigma-Aldrich, St Louis, MO, USA) plus antibiotics supplemented with 10% FBS. For adenoviral transduction experiments, mPAC cells were seeded onto 10 cm plates and treated one day later with Ad.Ngn3-GFP adenoviruses (purified) at a multiplicity of infection (moi) of 10 unless otherwise indicated O/N. Cells were harvested for FACS sorting ∼42 h after transduction. The bicistronic adenovirus encoding Ngn3-GFP was kindly provided by Dr. H. Heimberg^{48 (link)}.

Mouse insulinoma β-cell lines MIN6 (gift from Prof. Miyazaki, Osaka University, Osaka, Japan)^{30 (link)} and MIN6-m9 (gift from Prof. Seino, Kobe University, Kobe, Japan)^{31 (link)} were both cultured in DMEM (4.5 g/L glucose, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Biowest, Funakoshi Co., Ltd, Tokyo Japan), 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, Yokohama Japan), and 5 × 10⁻⁴% β-mercaptoethanol (Sigma-Aldrich). All cell cultures were incubated at 37 °C in 5% CO₂. Culture medium was changed every 2 days.
Before seeding in spheroid culture devices and as a control, both cell types were cultured in monolayer and were passaged by trypsinization (0.25% Trypsin-EDTA, Gibco, Thermo Fisher Scientific) once a week with a split ratio of 1:4. Seeding cell density for both PDMS- and PMMA-chips was varied at a range from 500 to 3,000 cells/well during optimization, and 1,000 cells/well was chosen for further experiments.
For the experiment with antioxidants: L-Ascorbic Acid Phosphate Magnesium Salt n-Hydrate (AA2P, FUJIFILM Wako Laboratory Chemicals, Tokyo, Japan) concentration in culture medium was at a range of 0.04–0.5 mM; N-Acetyl-L-cysteine (NAC, Sigma-Aldrich) was at a range of 1–10 mM; and DTT (Funakoshi, Co., Ltd., Tokyo, Japan) was at a range of 0.1–1 mM.

Human neuroblastoma SH-SY5Y cells were cultured in DMEM 4.5 g/L glucose (Sigma Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Sigma Aldrich, St. Louis, MO), 2 mM l-glutamine (Corning, Manassas, VA), 100 UI/mL penicillin and streptomycin solution (Corning, Manassas, VA) and 10.000 U/mL amphotericin B (antimycotic solution) (Sigma Aldrich, St. Louis, MO) in a 5% CO2 humidified atmosphere, at 37°C. Cells were maintained in 75 cm² flasks at a concentration of 2 × 106 cells/mL by passage every two to three days. To induce oxidative stress, cells were treated with H2O2 (Carlo Erba, Milan, Italy) at a final concentration of 100 μM for 1 h, followed by 24 h of recovery in EVs-depleted medium. After performing the treatments, cells were harvested for further analyses.

MSCs were seeded in Dulbecco modified Eagle’s medium (DMEM, 4.5 g/L glucose, Sigma-Aldrich) with 1 μM DEX, 0.5 mM indomethacin, 0.5 mM 3-isobutyl-1- methylxanthine, 10% FBS, 1% (v/v) P/S, 10 μg/mL insulin (Sigma-Aldrich) for 3 weeks. Adipogenesis was evaluated by Oil Red O staining. A stock solution was prepared from Oil Red O (0.5%) and isopropanol (Sigma-Aldrich). To prepare the working solution, 6 mL of the stock solution was mixed with 4 mL of distilled water and kept for 1 h at room temperature (23°C). The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min. Fixed cells were stained with the working solution for 20 min, and then rinsed with PBS. Stained cells were incubated with absolute isopropanol for 15 min, and optical density (OD) was determined at 520 nm [22 ].

Human primary pancreatic cancer cell lines (MaPac107 and PaCaDD159. PaCaDD165) were originally derived from patient tumour tissues [102 (link)] (Table 2). The cells were cultured in Dresden medium [6 (link)] consisting of Dulbecco’s Modified Eagle Medium (DMEM, 4.5 g/L glucose, Sigma-Aldrich, Taufkirchen, Germany) and Keratinocyte serum-free medium (KSFM, Thermo Fisher Scientific, Waltham, MA, USA) at a ratio of 2:1. DMEM was supplemented with 1% penicillin/streptomycin (P/S; Sigma-Aldrich, Taufkirchen, Germany) and 20% foetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). KSFM was supplemented with human recombinant epidermal growth factor (rEGF) and bovine pituitary extract (BPE). The cells were maintained in an atmosphere of 5% CO₂ at 37 °C and passaged at a 1:3 ratio. The cell culture medium was changed every two days.