Development of Complex Liver Organoids Using Cell-Specific Patterned Biomaterials

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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.