U bottom cell repellent plates

14TF iENDO cells were allowed to form 3D organoids by plating 25,000-iENDO cells/well in low attachment 96 well U bottom cell repellent plates (Cat. No.650970, Greiner-bio one, Belgium) by spinning at 300g for 5 min in Liver Differentiation medium (LDM) consisting of 60% DMEM low glucose; 40% MCDB-201; 1x-Penicillin-Streptomycin; (10,000 U/mL)); 0.25x LA-BSA (100x); 0.25x ITS-A (100x); 100nM, L-ascorbic Acid; 1μM dexamethasone; 50μM, 2-mercaptoethanol (50mM) supplemented with in addition as follows, for protocol-A, day 0 to 4: 50 ng/ml aFGF (Cat.No. 100-17A, Peprotech, NJ, USA) with or without 0.6% DMSO (Cat No.D2650 Sigma-Aldrich, Saint Louis, MO, USA) and, subsequently, from day 4 to 20 with 20 ng/ml HGF (Cat No.100-39H, Peprotech, NJ, USA) with or without 2% DMSO. For protocol B, organoids were cultured in 20 ng/ml HGF and 2% DMSO from day 0 to 16. Compacted organoids were maintained in a 37°C, 21% O₂, 5% CO₂ incubator, with medium change every 2 days until day 20 and 16 (protocol-A and B respectively). On the last day of differentiation organoids were collected for qRT-PCR. For immunostaining, organoids were fixed with 4% PFA overnight at 4°C and subsequently, stored in 70% ethanol at 4°C until embedding. Differentiation medium was collected and stored at -80°C for albumin ELISA.