3D Printing and Extracellular Matrix Culture Strategies for Liver and Intrahepatic Biliary Tree Tissue Engineering

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Currently, the only treatment for end stage liver disease is transplantation, of which there is a critical shortage of available donor tissue. Recent developments in 3D printing have greatly accelerated progress in the field of liver tissue engineering. A variety of 3D printing and additive manufacturing techniques show great promise in tissue engineering and basic science realms, the most promising of which is direct extrusion of cell-laden hydrogels. Despite these advances, investigations into 3D printing for recreation of the macro- and microstructural components of the liver are still in their infancy. Tailored biomaterials need to be patterned in biologically inspired geometries to induce tissue formation. Here, I will present several strategies that harness 3D-printed geometry, biomaterial bioactivity, or both, in order to influence liver tissue formation and function in vitro.', 'The creation of uniform and geometrically repetitive tissue scaffolds with 3D-printed gelatin enables control over aggregation of cells and nutrient diffusion. When seeding a human hepatocyte cell line onto scaffolds of different geometries, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin scaffolds compared to a less tortuous geometry and to 2D controls. Gelatin, however, is a highly processed and minimally bioactive material. Other biomaterials need to be investigated for their potential in liver tissue engineering.', 'Liver decellularized extracellular matrix (dECM) hydrogels have several advantages, primarily tissue-specific bioactivity. I sought to apply liver dECM hydrogels to an understudied aspect of liver tissue engineering: the intrahepatic biliary tree. The finer branches of the biliary tree are structurally and functionally heterogeneous. Here I demonstrate the ability of liver dECM hydrogels to induce the in vitro formation of complex biliary networks using encapsulated mouse biliary epithelial cells (cholangiocytes). This phenomenon is not observed with collagen gels or Matrigel. I also illustrate phenotypic stability as well as preliminary function of cholangiocytes, evident by polarization and transporter activity. To better define the mechanism of duct formation, I utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.', 'However, dECM hydrogels display poor printability on their own, and necessitate additives or support structures to enable true 3D fabrication. I show that a sacrificial material serve as a platform into which dECM hydrogel can be co-printed. Varying several aspects of 3D-printed strut geometry, such as strut width and angle, can control the formation of biliary trees, confirmed with computational 3D image analysis. This system also enables fabrication of a true multi-layer dECM structure and the formation of 3D biliary trees into which other cell types can be seeded. Furthermore, we show that hepatocyte spheroids can also be easily incorporated within this system, and that the seeding sequence influences the resulting structures after prolonged culture.

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  • 10/08/2019
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