Multilayered and heterogeneous hydrogel construct printing system with crosslinking aerosol

Microextrusion bioprinting has been used to recreate the complex architecture and composition of a physiological system through the quick and accurate handling of various biomaterials. However, existing techniques are limited in precisely fabricating complex constructs, including multilayers and heterogeneous patterns with distinct regions, because the extruded bioink spreads rapidly upon contact with the substrate and is partially mixed with subsequently printed bioinks. This issue leads to difficulties in accurately and stably constructing multi-material structures with clear interfaces for prolonged printing before gelation. To fabricate multilayered and heterogeneous constructs, a bioprinting system should be able to continuously extrude various biomaterials and simultaneously crosslink the extruded bioink to stabilize the printed construct. In this study, a multiple-bioink printing system was developed by integrating a multibarrel nozzle for extruding multiple bioinks with a nebulizer for simultaneous crosslinking. The crosslinking aerosol sprayed from the nebulizer was able to gelate the various hydrogel bioinks as they were extruded through the multibarrel nozzle. Such aerosol-based crosslinking improved printing resolution and stability. The developed bioprinting system showed the possibility of recapitulating the physiological complex architecture such as a cancer microenvironment with well-defined interfaces between regions of different mechanical properties and cellular compositions. Using the integrated bioprinting system, a multilayered and heterogeneous construct was printed with four bioinks, including three types of cells (breast cancer cells, stromal cells, and vascular endothelial cells). The printed biological model was characterized by analyzing cancer cell migration and vascular network formation. The developed multiple-bioink printing system is expected to be highly efficient in recapitulating complex tissues and their environments with compartmentalized regions.