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Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into a pseudostratified epithelium. Further, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases. 

Abstract 3D bioprinting additively assembles bio‐inks to manufacture tissue‐mimicking biological constructs, but with the typical building blocks limited to 1D filaments. Here, it is developed a voxelated bioprinting technique for the digital assembly of spherical particles (DASP), which are effectively 0D voxels—the basic unit of 3D structures. It is shown that DASP enables on‐demand generation, deposition, and assembly of viscoelastic bio‐ink droplets. A two‐parameter diagram is developed to outline the viscoelasticity of bio‐inks required for printing spherical particles of good fidelity. Moreover, a strategy is developed for engineering bio‐inks with independently controllable viscoelasticity and mesh size, two of the most important yet intrinsically coupled physical properties of biomaterials. Using DASP, mechanically robust, multiscale porous scaffolds composed of interconnected yet distinguishable hydrogel particles are created. Finally, it is demonstrated the application of the scaffolds in encapsulating human pancreatic islets for sustained responsive insulin release. Together with the knowledge of bio‐ink design, DASP might be used to engineer highly heterogeneous, yet tightly organized tissue constructs for therapeutic applications.