Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung.

extrusion bioprinting is mainly used for constructing volumetric structures in a layer-wise manner. [5] Although the layerby-layer bioprinting method is functional in majority of the cases, [6] there are limitations associated with creating anisotropic tissues, such as muscle fibers [7,8] and nerve fibers [9] that heavily rely on cellular alignment for their physiologies. Therefore, developing a versatile strategy that allows convenient 3D bioprinting synergized with simultaneous generation of structural anisotropy is essential for these applications.Numerous studies have shown that porous hydrogel scaffolds can potentially enhance cell spreading and proliferation. [10,11] In particular, ice-templating, one of the most widely utilized techniques for the fabrication of materials with anisotropic microchannels, allows control over pore morphologies by controlling directional ice formation in a suspension of solute(s). [12][13][14][15] During the freezing process, ice crystals form and propagate through a set direction within the biomaterial solution. When the construct cross-links and thaws, the melted ice crystals form interconnected anisotropic microchannels within the scaffold. Importantly, previous studies have clearly demonstrated that the presence of anisotropic microchannels enhances Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.