Mechanical cues promote HepaRG cell-based hepatic organoid maturation via focal adhesion and YAP-dependent signaling

Lack of functional hepatocytes poses challenges for drug safety testing and therapeutic applications. Biomechanical cues are known to potentiate differentiation of liver stem cells into hepatocyte-like cells when combined with biochemical factors that regulate hepatocyte maturation. Here we aimed to generate mature hepatic organoids (HepaRG cells-based) using a three-dimensional bioreactor, the Rotating Flat Chamber (RFC). Results demonstrated that hepatic organoids maintained high viability during long-term culture in RFC. Expressions of hepatocyte-specific markers of albumin (ALB), cytokeratin 18 (CK18), CYP3A4, and CYP1A2 were significantly higher but β1-integrin-FAK-talin-vinculin signaling was remarkably prohibited in RFC-cultured organoids compared to static controls. Typical functions of glycogen synthesis, indocyanine green excretion, and low-density lipoprotein uptake were also markedly enhanced in RFC. Notably, RFC culture significantly increased cytoplasmic localization of YAP protein, promoting differentiation and maturation of hepatic organoids. These findings indicated that rotating culture provided favorable environment for hepatocyte maturation than conventional culture with downregulation of β1-integrin-related mechanotransduction.