Abstract
In this study, we present the development of a cryobioink designed to fabricate anisotropic scaffolds that support both neural and muscle cell alignment. Given the critical role of cellular organization in nerve fibers and neuromuscular junctions, we employed a vertical cryobioprinting-enabled ice-templating technique to create scaffolds with aligned microchannels. These channels facilitated cell alignment, which is important in modeling neural and neuromuscular tissues. By integrating hyaluronic acid-methacrylate (HAMA) with gelatin methacryloyl and the necessary cryoprotective agent melezitose, we showcased that the cryobioink could preserve cell viability during freezing/thawing processes, even at low temperatures employed during cryobioprinting. We optimized HAMA concentration to enhance neural cell viability and alignment, and successfully constructed anisotropic scaffolds featuring distinct sections that contained muscle and neural cells, establishing a model for neuromuscular junctions. The resulting models provide a versatile platform for studying nerve fibers and neuromuscular dysfunctions, offering potential advancements in neural regeneration research.