The growth medium for the flat and dECM inserts was refreshed 6-18 hours after seeding, and then every 2-3 days over 7 days of proliferation. To start differentiation, the growth medium was replaced with 1 mL of 50% growth medium and 50% differentiation medium composed of HepaRG differentiation medium supplement with antibiotics (ADD711C, Biopredic International, Saint-Gregoire, France) in Williams E medium with GlutaMAX supplement (32551020, Thermo Fisher, Pittsburgh, PA, USA). Afterward, the medium was replaced with 1 mL differentiation medium every 2-3 days.