Rapid Extrusion of Myotube-Scale Alginate Fibers for Structured Cultivated Meat

Cultivated meat (CM) scaffolds must be edible and produced with high throughput. This study introduces a novel approach for structured CM production using alginate-based scaffolds prepared using a liquid-liquid extrusion method called Pressurized Apparatus for Striated Tissue Assembly (PASTA). We hypothesized that PASTA scaffolds could enable the creation of tissue constructs exceeding 500 µm thickness with food-scale throughput. Leveraging sub-boiling hydrothermal degradation, we modulated the biophysical properties of alginate, resulting in controlled reductions in viscosity, zeta potential, and hydrogel mechanical properties. PASTA extrusion consistently produced hydrogel fibers 10-42% smaller than the extrusion outlet diameter. Murine C2C12 myoblasts cultured on PASTA scaffolds modified with adhesive peptides adhered, proliferated, and differentiated along the noodle axis. This was confirmed by a 3.5-fold increase in DNA quantity over 4 days, substantial cell viability after 5 days in culture, late-stage myogenic gene expression (Myog, Myh2), and positive immunohistochemistry for myosin heavy chain. PASTA enables rapid, parallelizable production of aligned fiber scaffolds with physiologically relevant dimensions using inexpensive, food-grade alginate. These findings demonstrate a proof-of-concept scalable and modular platform to produce cell-laden tissues for manufacturing structured CM.