Microtissue geometry and cell-generated forces drive patterning of liver progenitor cell differentiation in 3D

Towards understanding the impact of mechanical signaling on progenitor cell differentiation in three dimensional (3D) microenvironments, we implemented a hydrogel based microwell platform to produce arrays of multicellular microtissues in constrained geometries, which cause distinct profiles of mechanical signals. We applied this to a model liver development system to investigate the impact of geometry and stress on early liver progenitor cell fate. We fabricated 3D liver progenitor cell microtissues of varied geometries, including cylinder and toroid, and used image segmentation to track individual single cell fate. We observed patterning of hepatocytic makers to the outer shell of the microtissues, except at the inner diameter surface of the toroids. Biliary markers were distributed throughout the interior regions and was increased in toroid tissues compared to cylinder tissues. Finite element models of predicted stress distributions demonstrated that cell-cell tension correlated with hepatocytic fate, while compression correlated with decreased hepatocytic and increased biliary fate. This combined approach integrating microfabrication, imaging and analysis, and mechanical modeling demonstrate of how microtissue geometry can drive patterning of mechanical stresses that regulate cell differentiation trajectories. It also can serve as a platform for the further investigation of signaling mechanisms in the liver and other systems.