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，常导致肝硬化，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，由于缺乏胆管运输系统，难以准确地模拟肝脏疾病。含有胆管的肝类器官很难制造，因为肝脏中的肝细胞和胆管细胞对Notch信号和生物力学线索的需求截然不同，而目前的3d培养技术无法同时提供这些信息。因此，迫切需要一种能够同时向共培养的两种类型的肝细胞传递靶向Notch信号和生物力学线索的技术，以维持它们的成熟。令人兴奋的初步研究表明，基于双生物墨水的生物打印结构可以在共培养基质中提供细胞类型特异性信号。此外，在他们之前工作的基础上，PI已经开发出新的工程矩阵，以细胞特异性的方式精确调整生物力学线索（刚度和粘弹性），这支持了3D人类胆管网络的生长。因此，本研究的目的是评估和优化Notch信号和生物机械信号在共培养肝细胞中发育具有综合胆流系统的类肝器官的作用。其基本原理是，具有综合胆汁流动系统的肝类器官将模拟肝脏功能，从而改善疾病建模和药物测试。本研究将有两个具体目标：(1)确定Notch时空激活对肝类器官功能的影响；(2)通过细胞类型特异性的生物力学线索优化肝类器官的成熟。在第一个目标中，生物打印构建体将使用两种不同的，带有或不带有光激活Notch配体的细胞负载生物墨水来开发，以在共培养中实现靶向Notch激活。类器官将使用Notch靶基因和一系列肝功能检测进行分析。在第二个目标中，将开发一个18条件的基质筛选，以系统地评估和优化生物力学线索对生物打印共培养构建中的肝类器官的影响。最后，将在共培养构建中评估机械感知机制。这项拟议的研究预计将具有重要意义，因为它将利用靶向Notch信号和生物力学线索，为具有治疗应用的集成胆管的肝类器官的开发提供信息，并将培养生物打印、生物材料和肝组织工程领域的多样化本科生。

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.