Development of Biomimetic Edible Scaffolds for Cultured Meat Based on the Traditional Freeze-Drying Method for Ito-Kanten (Japanese Freeze-Dried Agar)

In this study, we aimed to develop soy protein-derived edible porous hydrogel scaffolds for cultured meat based on mechanical anisotropy to mimic the physical and biochemical properties of muscle tissues. Based on the traditional Japanese <i>Ito-Kanten</i> (thread agar) freeze-thaw process, we used liquid nitrogen directional freezing combined with ion crosslinking to fabricate an aligned scaffold composed of soy protein isolate (SPI), carrageenan (CA), and sodium alginate (SA). SPI, CA, and SA were dissolved in water, heated, mixed, and subjected to directional freezing in liquid nitrogen. The frozen gel was immersed in Ca²⁺ and K⁺ solutions for low-temperature crosslinking, followed by a second freezing step and lyophilization to create the SPI/CA/SA cryogel scaffold with anisotropic pore structure. Furthermore, C2C12 myoblasts were seeded onto the scaffold. After 14 d of dynamic culture, the cells exhibited significant differentiation along the aligned structure of the scaffold. Overall, our developed anisotropic scaffold provided a biocompatible environment to promote directed cell differentiation, showing potential for cultured meat production and serving as a sustainable protein source.