Speaker
Description
The structural alignment of cells is a hallmark of functional neural and neuromuscular tissues. However, replicating this anisotropic architecture in engineered three-dimensional (3D) constructs remains a significant challenge in tissue engineering. In this study, we introduce a cryobioprinting strategy capable of fabricating aligned, multi-cell-type scaffolds for modeling neural tissues and neuromuscular junctions (NMJs).
We developed a hyaluronic acid methacrylate (HAMA)-based cryobioink, incorporating gelatin methacryloyl (GelMA) and the cryoprotectant melezitose, to maintain high cell viability during the extreme freezing and thawing conditions of cryobioprinting and to improve the biomimetic aspect of the neural tissue model. Utilizing a vertical cryofabrication process, we engineered anisotropic scaffolds featuring aligned microchannels that guide the spatial orientation of embedded neural and muscle cells.
The cryobioink formulation was optimized to maximize post-printing cell survival. Constructs fabricated with the optimized bioink supported up to 95% viability for neural cells, with similarly high viability observed for myoblasts after seven days in culture. Unlike traditional methods where neural cells are seeded onto scaffolds post-cryofabrication, our approach enables direct encapsulation and alignment of cells during the printing process, allowing for precise spatial positioning within the scaffold.
The microchannel size was tunable by adjusting freezing temperatures and cryoprotectant composition. Following co-culture and differentiation, the constructs exhibited not only sustained high cell viability and alignment, but also functional features of NMJs, such as acetylcholine receptor clustering at the muscle–neuron interface.
Furthermore, we utilized a vertical cryobioprinting setup with coaxial nozzles to fabricate complex, multi-segmented structures. This configuration enabled the creation of freestanding constructs featuring distinct bioink compositions and consistently aligned microchannels across each segment, providing precise spatial control and enhanced structural design.
Beyond demonstrating a robust method for fabricating anisotropic neural and neuromuscular tissue constructs, this work highlights hyaluronic acid’s role as a functional cryoprotectant. The cryo-printed constructs also exhibited enhanced resistance to swelling and degradation, which is crucial for preserving the fidelity and long-term integrity of the anisotropic scaffold.
In summary, our cryobioprinting approach and optimized HAMA-based bioink provide a versatile and effective platform for creating 3D neural and neuromuscular tissue models with structural and functional relevance (Figure 1). This strategy paves the way for the development of shelf-stable, anisotropic tissue constructs for applications in disease modeling, drug screening, and regenerative medicine.
74734105346