Low-temperature anisotropic embedded 3D bioprinting of shear-oriented porous GelMA bioink

• A feasible strategy for developing anisotropic bioinks based on phase-shear alignment principles. • Cooling‑based anisotropic embedded 3D bioprinting platform using a temperature‑inert support bath. • Aligned microstructures in printed cell-loaded patches effectively guide cell orientation. Structural anisotropy is essential for tissue function, enabling directional biological processes such as contraction and mechano-transduction. Conventional 3D bioprinting often fails to fabricate stable, cell-laden constructs with sustained structural anisotropy. Here, a low-temperature embedded 3D bioprinting strategy is presented that leverages the viscosity difference between PEO and GelMA to induce phase separation and shear alignment, resulting in microstructures with controlled porosity and orientation. The temperature-inert supporting bath is used, which provides favorable shear-thinning behavior below 37 °C for bioink stabilization and transitions into gradual dissolution upon warming above 37 °C for removal. Reversible hydrogen-bond networks of GelMA formed under low-temperature conditions enable long-term stabilization of the aligned microstructures, preventing structural disorganization. Subsequent photo-crosslinking ensures permanent stabilization of the anisotropic microstructures. Following the removal of the PEO and support bath, a porous aligned microstructure is obtained for enhancing permeability. The fabricated C2C12-encapsulated patch not only exhibited pronounced directional elongation but also showed a more than 3-fold increase in Myogenin expression compared to isotropic controls. This approach enables precise control of microstructural anisotropy without external fields or specialized ink formulations. By utilizing phase‑separation‑induced shear alignment, this low-temperature anisotropic embedded bioprinting strategy facilitates the fabrication of anisotropic constructs, advancing functional artificial tissue engineering.