Global seafood demand continues to increase, raising concerns over resource depletion and environmental impact. As a sustainable alternative, cell-cultivated fish requires edible scaffolds that support cell adhesion and tissue formation. This study developed a hierarchical 3D porous scaffold using fish gelatin/chitosan microfibers (FG/C-MF) via Pickering emulsion templating and 3D printing to enhance structural and biological performance.

Fibrous scaffolds were ground into fine FG/C-MF particles and used to prepare Pickering emulsion ink. Particle morphology was examined by SEM, and their wettability and emulsifying ability were evaluated through contact angle measurements. The pH-dependent aggregation behavior was analyzed using zeta potential and optical microscopy. Since FG/C-MF tended to collapse after freeze-drying due to weak interparticle bonding, transglutaminase (TGase) crosslinking was applied to enhance structural integrity. The emulsion stability index (EI) before and after TGase treatment and the internal morphology under varying oil fractions were analyzed by SEM. Rheological measurements were conducted to evaluate printability, and both molded and 3D-printed scaffolds were compared in terms of compressive strength and cell adhesion. Cell viability and distribution were visualized using Live/Dead staining.

The fibrous scaffolds were successfully converted into microfibrous particles, and stable suspensions were obtained by pH adjustment, which suppressed particle aggregation. TGase crosslinking markedly improved structural stability after freeze-drying. The internal pore size and interconnectivity varied depending on the oil fraction, leading to differences in porosity, mechanical integrity, and cell affinity. Rheological analysis confirmed that FG/C-MF-based Pickering emulsion ink exhibited viscoelasticity suitable for 3D printing. The printed scaffolds showed greater compressive strength and cell adhesion than molded ones, and Live/Dead staining demonstrated uniform cell attachment and high viability.

This study introduces a Pickering emulsion–based 3D printing strategy that enables tunable pore architecture and enhanced stability, advancing hierarchical porous scaffold technology for cultured seafood production.