A simple suspension culture method for generating human iPSC-derived liver organoids

Abstract Human liver in vitro models are indispensable for toxicity studies requiring metabolic competence. However, widely used two-dimensional hepatocyte-derived cell lines exhibit limited drug-metabolizing capacity, whereas primary human hepatocytes are constrained by limited availability, donor variability, dedifferentiation, and poor long-term functional stability. Although three-dimensional liver models offer greater physiological relevance, many existing methods depend on exogenous extracellular matrix (ECM) scaffolds or specialized equipment, thereby limiting standardization, scalability, and broader implementation. Here, we developed a simple, scalable suspension culture method for generating human induced pluripotent stem cell (iPSC)-derived liver organoids (HLOs) without exogenous ECM scaffolds or specialized equipment. The resulting HLOs secreted albumin and exhibited hepatic gene expression profiles by RNA-seq analysis. Notably, cytochrome P450 3A4 activity was maintained more stably over an extended culture period than under the ECM-embedding condition. Immunostaining further confirmed the presence of hepatocyte-like populations together with CK19-positive epithelial structures exhibiting E-cadherin-positive cell–cell adhesion and ZO-1-positive tight junction-associated regions. These findings indicate that the HLOs recapitulate key structural and functional features of human liver tissue. Collectively, our results demonstrate that this simple suspension culture method provides a practical and accessible strategy for generating HLOs with sustained hepatic functionality. This platform should facilitate broader application of HLOs in drug screening, toxicity assessment, disease modeling, and other in vitro settings requiring reproducible and scalable human liver models.