Engineering multiple Cell Co-Culture System with Fucoidan and Beeswax Hydrogels for Cultured Meat Applications

The co-culture of multiple cell types is critical for replicating native muscle complexity in cultured meat production. However, nutrient competition and divergent signaling among cell populations pose major challenges to in vitro tissue formation. This study investigates fucoidan (Fu) and beeswax-based alginate hydrogels (Bw/Algi) as potential scaffolds to address two key challenges: creating a supportive environment for multiple relevant cell types and promoting the formation of muscle-like tissue. We present an indirect 2D and 3D co-culture of C2C12 myoblasts, human endothelial cells (HECs), and mouse embryonic fibroblasts (MEFs), supported by Bw/Algi and Fu/Algi hydrogels. The Bw/Algi hydrogel supported cell adhesion with a pore size increased to 77.57% ± 7.88 versus 54.16% ± 4.64 in Bw. C2C12 cells reached confluency within five days without cytotoxicity. The Fu/Algi hydrogel (20 µg/mL Fu) supported 3D myogenic differentiation, myotube formation (area: 1,494,119 µm² ± 404; length: 20,723.01 µm ± 68). The engineered tissue had 9.825% protein versus 15.875% in native muscle. Thermal analysis confirmed structure retention during cooking. This co-culture system provides a promising platform for functional muscle-like tissue development in cultured meat. While the present study employs well-established model cell lines (C2C12, MEFs, and HECs), these results provide foundational insights into scaffold performance that will guide future validation with primary cells derived from edible species.