The liver has complex microenvironments, where parenchymal hepatocytes and non-parenchymal cells coexist. Hepatocytes exhibit different metabolic functions depending on their location by the oxygen gradient and the transcriptional changes of genes, which is called liver zonation. Three-dimensional (3D) liver tissue engineering has reproduced the complex microenvironments, but there is a limitation in analyzing them by location. In this study, a novel 3D tissue-level hepatic cell culture platform is developed via stacking the manipulable collagen sheets to spatially analyze the reconstructed metabolic zonation. The controlled assembly of the sheets containing hepatocytes and endothelial cells, respectively, creates a 3D co-culture environment that improves hepatic function. In addition, the sheet micropatterning can be used to control the accessibility of oxygen and nutrients in the stacked sheets. The disassembly of the stacked sheets enables a layer-by-layer analysis and allows us to confirm the metabolic zonation qualitatively. A demonstration of acetaminophen-induced liver injury using the stacked sheets shows the improved drug sensitivity by co-culture and chemical induction and presents the quantitative results of the different cellular responses to the drug by layers according to metabolic zonation. Therefore, this platform is expected to be used for an in-depth analysis of drug toxicity in complex tissues via spatial analysis.

肝脏具有复杂的微环境，实质肝细胞和非实质细胞共存。肝细胞根据氧梯度和基因的转录变化（取决于肝区带）的位置，表现出不同的代谢功能。三维（3D）肝组织工程学已经复制了复杂的微环境，但是按位置分析它们存在局限性。在这项研究中，通过堆叠可操作的胶原片来空间分析重建的代谢区带，从而开发了一种新颖的3D组织级肝细胞培养平台。分别包含肝细胞和内皮细胞的薄片的受控组装产生了改善肝功能的3D共培养环境。此外，薄板微图案化可用于控制堆叠薄板中氧气和养分的可及性。堆叠板的拆卸可进行逐层分析，并允许我们定性确定代谢区带。使用堆叠纸对乙酰氨基酚引起的肝损伤的演示显示了通过共培养和化学诱导提高的药物敏感性，并根据代谢分区逐层显示了不同细胞对药物反应的定量结果。因此，该平台有望用于通过空间分析对复杂组织中的药物毒性进行深入分析。使用堆叠纸对乙酰氨基酚引起的肝损伤的演示显示了通过共培养和化学诱导提高的药物敏感性，并根据代谢分区逐层显示了不同细胞对药物反应的定量结果。因此，该平台有望用于通过空间分析对复杂组织中的药物毒性进行深入分析。使用堆叠纸对乙酰氨基酚引起的肝损伤的演示显示了通过共培养和化学诱导提高的药物敏感性，并根据代谢分区逐层显示了不同细胞对药物反应的定量结果。因此，该平台有望用于通过空间分析对复杂组织中的药物毒性进行深入分析。