Development of Complex Liver Organoids Using Cell-Specific Patterned Biomaterials

肝胆管疾病是肝移植的主要原因之一，经常导致肝硬变，影响数百万美国公民。目前人类肝脏类器官缺乏完整的胆管，由于缺乏胆汁转运系统，因此很难准确地建立肝病模型。含有胆管的肝脏类器官很难创造，因为肝脏中的肝细胞和胆管细胞对Notch信号和生物力学信号的需求截然不同，而目前的3D培养技术无法同时提供这两种信号。因此，迫切需要一种技术，能够在共培养中同时向这两种类型的肝细胞传递靶向Notch信号和生物力学信号，以保持它们的成熟。令人兴奋的初步研究表明，基于双生物墨水的生物打印构建体可以在共培养基质中提供特定细胞类型的信号。此外，在他们以前工作的基础上，PI开发了新的工程基质，以细胞特有的方式精确调整生物机械线索(硬度和粘弹性)，支持3D人体胆管网络的生长。因此，这项建议的目的是评估和优化在共培养的肝细胞中图案化的Notch信号和生物机械线索的作用，以发展具有整合胆汁流动系统的肝脏有机物质。其基本原理是，具有集成胆汁流动系统的肝脏有机化合物将模拟肝功能，因此将改进疾病建模和药物测试。拟议的研究将追求两个具体目标：(1)确定时空Notch激活对肝脏类器官功能的影响，以及(2)通过特定细胞类型的生物力学线索优化肝脏类器官的成熟。在第一个目标中，生物打印构建体将使用两种不同的、细胞负载的生物墨水开发，带有和不带有可光激活的Notch配体，以在共培养中实现有针对性的Notch激活。这些有机化合物将使用Notch靶基因和一系列肝功能分析进行分析。在第二个目标中，将开发一个18个条件的矩阵筛选，以系统地评估和优化生物打印的共培养构建中图案化的生物力学线索对肝脏器官的影响。最后，对共文化建构中的机械感知机制进行了评价。预计这项拟议的研究将具有重要意义，因为它将利用有针对性的Notch信号和生物力学提示，为治疗应用提供具有整合胆管的肝脏器官类化合物的开发信息，并将在生物打印、生物材料和肝组织工程领域培训不同的本科生群体。

Liver bile-duct diseases are one of the main causes of liver transplantation, and often result in liver cirrhosis, affecting millions of US citizens. Current human liver organoids lack integrated bile ducts, which makes it difficult to accurately model liver diseases due to a lack of bile-transport system. Liver organoids containing bile ducts have been difficult to create because the hepatocytes and bile-duct cells in the liver have vastly different needs for both Notch signaling and biomechanical cues, which the current 3D-culture techniques are not capable of providing simultaneously. Thus, there is a critical need for a technology that can simultaneously deliver targeted Notch signaling and biomechanical cues to both types of liver cells in co-culture, in order to maintain their maturation. Exciting preliminary studies indicate that the dual-bioink-based bioprinted constructs can provide cell-type-specific signals within a co-culture matrix. Moreover, building on their previous work, the PI has developed new engineered matrix to precisely tune the bio-mechanical cues (stiffness and viscoelasticity) in a cell-specific manner, which supports the growth of 3D human bile-duct network. Accordingly, the objective of this proposal is to evaluate and optimize the effect of patterned Notch signaling and bio-mechanical cues in co-cultured liver cells to develop liver organoids with integrated bile-flow system. The rationale is that liver organoids with integrated bile-flow system will mimic liver function, thus will improve disease modelling and drug testing. The proposed research will pursue two specific aims: (1) Determine the effect of spatio-temporal Notch activation on liver organoid functions, and (2) Optimize the maturation of liver organoid via cell-type-specific biomechanical cues. In the first aim, bioprinted constructs will be developed using two distinct, cell-laden bioinks with and without photo- activatable Notch ligands, to achieve targeted Notch activation in co-culture. The organoids will be analyzed using Notch target genes and a range of liver functional assays. In the second aim, an 18-condition matrix screen will be developed to systematically evaluate and optimize the effect of patterned biomechanical cues on liver organoids in a bioprinted co-culture construct. Finally, the mechano-sensing mechanism will be evaluated in co-culture construct. The proposed research is expected to be significant because it will leverage targeted Notch signaling and biomechanical cues to inform the development of liver organoids with integrated bile ducts for therapeutic applications, and will train a diverse group of undergraduate students in the area of bioprinting, biomaterials, and liver tissue engineering.