Construction of 3D modular hepatic tissue using micropatterned collagen sheet for tissue-level cytotoxicity assay

Abstract

The hepatic lobule, which is a structural and functional unit of the liver, has the complex microenvironments in which parenchymal hepatocytes and non-parenchymal cells coexist. Hepatocytes exhibit different functional levels depending on their location by the oxygen gradient and the transcriptional changes of genes, which is called liver zonation. The engineered liver tissues have reproduced the complex environments for physiologically-relevant in vitro drug testing, but there was limited in analyzing them by location. Here, we present a novel three-dimensional (3D) tissue-level hepatic cell culture platform layering the manipulable collagen sheets in order to analyze the reconstructed liver zonation spatially. The developed micropatterned collagen sheet combined with paper support enabled the manipulation of an extracellular matrix-based sheet module and provided the 3D microenvironments for various cell types. The assembly of the sheets containing hepatocytes and endothelial cells, respectively, created a 3D co-culture environment, which improved hepatic function, and the layer arrangement of the stacked sheets also affected the function. In addition, the sheet micropatterning was able to control the accessibility of oxygen and nutrients in the stacked sheets. The disassembly of the stacked sheets enabled layer-by-layer analysis and allowed to confirm the hepatic zonation qualitatively. A demonstration of acetaminophen-induced liver injury using the stacked sheets showed the improved drug sensitivity with co-culture and chemical drug-metabolic induction and presented the quantitative results of the different cellular responses to the drug by layer according to liver zonation. Therefore, this platform is expected to be used for in-depth drug toxicity assays of complex liver tissue via spatial analysis.