The MTS Assay Kit (ab197010, Abcam, Cambridge, UK) was used to measure the metabolic activity of differentiated HepaRG cells 13-43 days after differentiation. For each well of flat or dECM insert culture, the differentiation medium was replaced with 500 µL of phenol red-free differentiation medium + 50 µL MTS Reagent Solution. A blank with 20 µL MTS Reagent Solution added to 200 µL of phenol red-free differentiation medium was also prepared. The cell culture and blank were incubated at 37 °C for 2 hours, rocking at culture and blank were pipetted into a 96-well plate, and the absorbance was measured on a plate reader (SpectraMax i3x, Molecular Devices, San Jose, CA, USA) at 490 nm.