Coagulation-assisted extrusion 3D-printing of soy and pea protein isolate scaffolds for cultivated meat applications

: This work presents a coagulation-assisted extrusion 3D-printing and sequential dual-crosslinking strategy for producing mechanically stable, polysaccharide-free soy and pea protein scaffolds with microscale muscle-mimetic architecture for whole-cut cultivated meat. Current plant-protein scaffold fabrication methods remain limited by trade-offs between structural resolution, aqueous stability, and edible composition. Conventional extrusion-based 3D printing often produces millimeter-scale filaments, whereas coagulation-based fiber fabrication approaches frequently require polysaccharide reinforcement and offer limited architectural control. Here, soy protein isolate (SPI) and pea protein isolate (PPI) inks were directly extruded into a coagulation bath within a programmable 3D-printing platform, enabling rapid filament stabilization. This approach produced continuous 50–100 μm fibers, comparable to native muscle fibers, arranged into interconnected porous networks with 25–35% porosity. Scaffold stabilization was achieved using sequential dual crosslinking, where low-dose genipin treatment (0.1 mM) provided initial chemical crosslinking, followed by dehydrothermal processing to reinforce the protein network. This approach produced a mild pink coloration rather than the intense blue color associated with higher genipin concentrations, while improving scaffold handling, aqueous stability, and mechanical integrity. The resulting scaffolds exhibited compressive moduli of 3–15 kPa, within the stiffness range associated with early myogenic differentiation. The scaffolds supported C2C12 myoblast and primary bovine satellite cell attachment, spreading, proliferation, infiltration, and myogenic differentiation. Fluorescence and SEM imaging confirmed close cell–fiber interactions, broad cell distribution, and myosin heavy chain-positive myotube-like structures. Together, these results establish coagulation-assisted extrusion 3D printing combined with sequential dual crosslinking as a protein-centric strategy for generating controlled fiber formation and programmable scaffold patterning, mechanically stable, and cell-supportive edible scaffolds for whole-cut cultivated meat applications.