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，并且通常导致肝硬化，影响数百万美国公民。目前的人类肝脏类器官缺乏完整的胆管，这使得由于缺乏胆汁转运系统而难以准确建模肝脏疾病。含有胆管的肝脏类器官一直难以创建，因为肝脏中的肝细胞和胆管细胞对Notch信号传导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。因此，迫切需要一种技术，该技术可以同时向共培养的两种类型的肝细胞递送靶向Notch信号传导和生物力学线索，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构建体可以在共培养基质内提供细胞类型特异性信号。此外，在他们以前工作的基础上，PI开发了新的工程基质，以细胞特异性方式精确调节生物力学线索（刚度和粘弹性），这支持了3D人体胆管网络的生长。因此，本提案的目的是评估和优化模式化Notch信号传导和生物机械线索在共培养的肝细胞中的作用，以开发具有整合的胆汁流动系统的肝类器官。基本原理是具有集成胆汁流动系统的肝脏类器官将模拟肝功能，从而改善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）通过细胞类型特异性生物力学线索优化肝脏类器官的成熟。在第一个目标中，将使用具有和不具有可光活化的Notch配体的两种不同的载有细胞的生物墨水来开发生物打印的构建体，以在共培养中实现靶向Notch活化。将使用Notch靶基因和一系列肝功能测定来分析类器官。在第二个目标中，将开发一个18条件矩阵筛选，以系统地评估和优化生物打印共培养构建体中图案化生物力学线索对肝脏类器官的影响。最后，将在共培养构建中评估机械感测机制。这项拟议的研究预计将具有重要意义，因为它将利用有针对性的Notch信号和生物力学线索，为开发具有集成胆管的肝脏类器官提供信息，以用于治疗应用，并将在生物打印，生物材料和肝脏组织工程领域培养一批多样化的本科生。

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.