Direct-write printing for producing biomimetic patterns with self-aligned neurites

Abstract Various techniques have been introduced to produce artificial neural constructs with aligned architectures, which have shown significantly improved neural function and regeneration. However, the techniques used to fabricate sophisticated patterns with aligned neurites show some limitations. Herein, we developed a direct-write printing process capable of producing versatile biomimetic patterns with aligned neurites using multiple cell types. Fibrin-based bio-ink was prepared for patterning with neuronal cells. After printing fixed pillars at both ends, microfibers were fabricated between the pillars using PC-12 neuronal cell/normal human dermal fibroblast (NHDF)-laden bio-ink and a direct-write printer. After two weeks of differentiation, aligned neurites were induced by the contractile force of the printed cells. We found that this self-induced alignment improved PC-12 differentiation and that neurite alignment could be adjusted by controlling the NHDF and bio-ink concentration. The bundle of cell-laden microfibers also showed uniform formation of neurites and synapse-like structures. Finally, we demonstrated the usefulness of the printing process by fabricating a Y-shaped branch and six-layered pattern. The six-layered pattern mimicking cerebral cortex tissue was produced by precise printing of two different colored cells. These results indicate that versatile biomimetic neural constructs composed of multiple cell types can be produced by our new direct-write printing process.