Three-dimensional (3D) printing offers a promising approach for fabricating customized scaffolds in cultivated meat. However, challenges remain achieving sufficient mechanical stability using food-grade materials, ensuring micron-level precision, and maintaining low-temperature compatibility suitable for fish cells.

To address these limitations, a bioink was designed using a double-network crosslinking strategy. The first network was formed through hydrogen bonding and polymer entanglement among iota-carrageenan (IC) for low-temperature gelation, sodium alginate (SA) for viscosity control, and cellulose nanocrystals (CNC) for enhanced strength and printing stability. An additional ionic network was introduced using CaCO₃and glucono-δ-lactone (GDL), completing the double-network structure. In polysaccharide-based bioinks, this internal gelation approach is widely employed to achieve gradual and uniform crosslinking, overcoming the rapid and uneven gelation associated with caused by CaCl₂. However, CaCO₃–GDL system induces a gradual pH drop during crosslinking, which may compromise the viability of fish cells. Moreover, although CaCO ₃ – GDL internal gelation is widely used in biomedical applications, it has not been systematically examined in the context of cultured meat. Therefore, this study aims to define the optimal range of gelation rate, printability and cell viability, establishing fundamental criteria for developing edible and cell-compatible scaffolds for cultivated fish.

Different combination of SA, IC and CNC were formulated and screened to identify the minimum gelation concentration. Rheological tests were conducted to confirm whether the inks exhibited suitable viscoelasticity for extrusion printing. The printability was further evaluated by measuring the minimum extrusion pressure and assessing printing fidelity. To determine optimal crosslinking conditions, pH changes at different GDL concentrations were monitored, as a drop below pH 6 may affect fish cell viability. Time sweep tests were then performed to verify gelation behavior over time. Based on the selected GDL concentration, degradation tests were carried out to assess long-term structural stability. Finally, the cytocompatibility of the bioinks was examined using leachate solutions for cell viability assay.

Increasing SA content enhanced the storage modulus (G’), whereas higher IC ratios accelerated gelation. Rheological analyses exhibited pronounced shear-thinning behavior, indicating adequate viscoelasticity for extrusion printing. The printed constructs demonstrated moderate shape fidelity with the rheological findings. pH monitoring revealed a gradual decrease from 7.4 to 6.2 with increasing GDL concentration, suggesting a controllable internal gelation window applicable for cell compatibility. Time sweep measurements showed a faster rise in G’ at higher GDL levels, confirming accelerated crosslinking kinetics. The printed products maintained their structural integrity up to 14 days, and cell viability tests using the leachate indicated approximately 80% survival, validating the cytocompatibility of the developed bioink.

These findings demonstrate that CaCO ₃ – GDL based double-network bioink enables controlled gelation, stable printability, and favorable cytocompatibility, providing a practical foundation for developing edible and cell-compatible scaffolds in cultivated fish.