Three-dimensional bioprinting of 'histomimetic' liver construct using hepatic organoid as tissue building blocks

Hepatic organoids are potential building blocks for three-dimensional (3D) bioprinting of liver tissue models, as they mimic tissue-level cellular patterning and functions. However, traditional hydrogel-embedded organoid development methods are less dynamic and require harsh retrieval techniques that negatively affect organoid integrity, function, and tissue continuity within bioprinted constructs. In this study, we evaluated the utility of mesenchymal stem cells (MSCs) and a thermoresponsive culture substrate, poly (N-isopropyl acrylamide-co-glycidyl methacrylate) (NGMA), for liver organoid formation and easy retrieval, as well as the potential of such non-invasively harvested organoids for extrusion-based 3D bioprinting. Primary rat liver cells were seeded on MSC-laden NGMA substrate and cocultured for 72 hours under defined conditions. The cells self-organized to form viable and functional liver organoids that detached from the substrate as organoid sheets when exposed to low temperature. Moreover, the organoid sheets and organoid-forming cells were independently encapsulated in methacrylated gelatin bioink to produce liver tissue constructs via extrusion bioprinting. Compared to cell-laden constructs, the organoid-laden construct exhibited higher levels of liver-specific gene expression and tissue functions such as protein synthesis, ammonia detoxification, and drug metabolism. Histological analysis of the organoid-laden construct revealed 'histomimetic' cellular organization and the expression of tissue-specific markers. Overall, the MSC-laden NGMA substrate proved to be an ideal platform for generating intact liver organoids for 3D bioprinting of histomimetic liver tissue models. The methodology established here can be used to develop a reliable liver tissue model for drug testing, disease modeling, and regenerative therapy.