Fabrication of Multiscale Porous Hydrogel Scaffold for Air–Liquid Interface Culture of Epithelial Cells Using Nebulized CaCl2-Assisted Three-Step Cross-Linking Three-Dimensional Printing

Creating thick tissue constructs that can enable air-liquid interface (ALI) culture is of great interest for the culture of epithelial cells. This paper presents a method of using nebulized calcium chloride (CaCl2)-assisted three-dimensional printing with a sacrificial polyvinyl alcohol (PVA) porogen component for the fabrication of a thick, multiscale porous scaffold for ALI cell culture. The ink used for scaffold fabrication is composed of gelatin microgels, PVA, sodium alginate, and a continuous gelatin solution. This fabrication approach utilizes ionic in situ cross-linking of sodium alginate using nebulized CaCl2, in addition to gelatin microgel jamming-based physical cross-linking during printing and enzymatic cross-linking of gelatin-based scaffolds post-printing, to create a robust structure, incorporating three different cross-linking steps. Exposure to the nebulized CaCl2 increases the storage modulus of the gel from 150 Pa to over 700 Pa and increases printing fidelity significantly. After cross-linking, the PVA component is leached from the scaffold, creating a network of pores to enhance transport. Printed scaffolds are able to transport 10 kDa molecules, roughly analogous to standard protein sizes. For applications in cell culture, the printing approach described here is used to fabricate a thick cylindrical scaffold with multiscale porosity, with printed macropores present in all but the solid top layer and micropores throughout the entire scaffold. Cells cultured on such porous scaffolds maintain a high degree of confluence, with an 88.1% surface coverage, while being exposed to air for multiple days. To demonstrate potential use of this approach to create drug screening platforms, cells grown on the scaffolds are exposed to the cancer drug Ulixertinib, delivered via scaffold transport. These results demonstrate that the presented fabrication method can be used to make porous scaffolds for the creation of an ALI culture environment, an important step towards biomimetic ALI models for biomedical applications, including therapeutic screening.