For thin-layer 3D culture, BD Matrigel stock solution (BD Biosciences) was diluted with ice-cold ReN cell differentiation medium (1:10 dilution ratio) and then vortexed with the cell pellets for 20 seconds. The final cell concentration for the mixture was approximately 2 × 106 cells/ml. The cell/Matrigel mixtures were immediately transferred into either Optilux Black/Clear bottom 96-well plates (100 μl/each well, BD Biosciences) or 8-chamber well Lab-Tek II coverglass plates (200 μl/each well, Thermo Scientific, Rockford, IL, USA) using pre-chilled pipettes. The plates were incubated for 1 hour at 37°C to form thin layer (100–300 nm) 3D gels at the bottom of the plates and the media were changed. The 3D-plated cells were differentiated for 4–12 weeks depending on the experiments; media was changed every 3–4 days. For thick-layer 3D cultures, BD Matrigel solution was diluted with the same volume of the ice-cold ReN cell differentiation medium (1:2 dilution ratio) and vortexed with ReN cell pellets for 20 seconds. The final cell concentration for the mixture was approximately 1 × 107 cells/ml. 400 μl of the cell/Matrigel mixtures were immediately transferred
