Fabrication of Hepatic Spheroid Microarray Chip with Biomimetic Microarchitecture for Drug Hepatotoxic Evaluation

ABSTRACT Multicellular hepatic spheroids that recapitulate biomimetic cellular assembly and enhanced cellular interaction have become useful 3D culture models for liver tissue engineering and hepatotoxic drug screening. Despite progress, current strategies for generating heterogeneous spheroids with multiple cell types mostly depend on cell random aggregation, lacking a well‐defined architecture that mimics natural liver tissue structure. Here, a microarray chip with a triangular chamber arrangement is developed to the fabrication of heterogeneous spheroids comprising hepatocytes, stellate, and endothelial cells in a multilayer organization. The triple cell co‐culture spheroids formed by in sequence seeding above three types of cells, yielding structures with a hepatic plate‐like core orderly coated by stellate and endothelial shells. These structured spheroids exhibit an enhanced structural integrity and liver functions such as albumin, MRP2, and the CYP3A4 and CYP1A2 enzyme expression levels compared to randomly distributed spheroids. Screening of hepatotoxic drugs by the hepatic spheroid microarray show increased sensitivity compared to 2D cultures. This approach represents a significant advancement toward control over cell spatial distribution in spheroids, and offers a valuable structured spheroid model with multilayer cellular organization for regenerative medicine and drug hepatotoxic evaluation.