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Protocols for the propagation of liver organoids rely on culture conditions for the establishment and expansion of hepatic organoids, followed by a second culture environment that removes proliferative signals, inhibits ductal lineage commitment, and induces differentiation towards hepatocyte-like cells. During expansion, hepatic organoids express a ductal phenotype characterized by the expression of Krt19. However, upon differentiation, hepatic organoids downregulate ductal markers and exhibit albumin secretion, CYP3A activity, LDL uptake, and glycogen storage. Use our to generate mature and functional human liver organoids that are suitable for further experimentation or biobanking.