Engineering a Compartmentalized Multi-Cell Co-Culture Hydrogel System Using Beeswax/Fucoidan/Alginate for Cultured Meat Modeling

Objective: Developing foundational biomaterial platforms for cultured meat research requires 3D co-culture systems capable of supporting multiple relevant cell types in a spatially organized manner. This study aimed to establish a compartmentalized tri-culture hydrogel disc incorporating a lipid-containing barrier phase as a proof-of-concept in vitro model. Methods: Beeswax/alginate (Bw/Algi) hydrogels were fabricated and evaluated for morphology and cytocompatibility as a lipid-containing scaffold component. Fucoidan/alginate (Fu/Algi) hydrogels were prepared at varying fucoidan concentrations and screened to identify conditions compatible with C2C12 viability and early-stage differentiation. A composite beeswax/fucoidan/alginate disc (Bw/Fu/Algi) was then assembled by casting cell-laden Fu/Algi regions (myoblasts, fibroblasts, and endothelial cells), separated by Bw/Algi barrier layers and ionically crosslinked with CaCl2. Scaffold performance was assessed using standard assays for morphology, cytocompatibility, myogenic marker expression, protein production, and thermal stability. Results: Bw/Algi supported cytocompatible C2C12 attachment and growth, while Fu/Algi exhibited concentration-dependent effects on myogenic marker expression, enabling selection of an optimized fucoidan concentration for 3D assembly. The final Bw/Fu/Algi disc maintained viable compartmentalized tri-culture and supported indirect co-culture through spatial separation by the Bw barrier. Myogenic regions exhibited myogenic marker expression with measurable protein production, and differential scanning calorimetry confirmed structural stability under heating. Conclusion: This work establishes a Bw/Fu/Algi tri-culture disc integrating a lipid-containing barrier component with hydrogel-based myogenic compartments, providing a preliminary platform for multicellular in vitro modeling and scaffold design relevant to cultured meat research.