Heterogeneous liver tissues with biliary branching and vascular elements through organoid bioprinting

Liver is dynamic, heterogeneous, and each cell type acts in concert to regulate its function. In vitro morphogenesis is limited, and self-assembled biliary and blood vessels system are absent from manufactured liver tissues. The combination of bioprinting and organoid technique offers spatial and cellular control over three-dimensional (3D) organ tissue manufacturing, allowing to build liver tissues with self-assembled structure in vitro. We developed a high-throughput PDMS microwell platform (PMP) generating uniform and functional hepatic organoid building blocks (HOBBs) which displayed cellular crosstalk and self-assembled structure. For bioprinting process, we developed three-level temperature control system and new quadratic material, i.e., alginate-gelatin-collagen-laminin (AGCL) biomaterial, realizing reproducible construction of liver tissues with requisite cellular density. Under long-term differentiation, bioprinted liver tissues exhibited enhanced hepatobiliary function, intrahepatic bile duct networks and angiogenic potential. To investigate the regulatory mechanisms of multicellular crosstalk and self-assembled of HOBBS, biliary branching morphogenesis, biomimetic liver tissue formation, and angiogenesis of HABs, transcriptome analysis was performed. Overall design: For 2D culture, HepaRG cells and HUVECs were maintained in growth medium. Hepatic organoid building blocks (HOBBs) were generated by co-culturing HepaRG cells and HUVECs, and cultured in HepaRG growth medium. HABs were constructed by bioprinting HOBBs and cultured with differentiation media ( HepaRG growth medium supplemented with 0.5% DMSO) for 14 days. The samples for RNA-seq were collected in TriZol.