Acemannan/alginate composite hydrogels promote STO cell spreading and metabolic activity

Alginate-based hydrogels are widely used as biocompatible scaffolds. However, their limited cell-adhesive properties restrict their application in tissue engineering. This study aimed to fabricate acemannan/alginate (ACE/ALG) composite hydrogels and evaluate their cytocompatibility and ability to support STO cell spreading and metabolic activity. Composite hydrogels were prepared by blending different concentrations of ACE with alginate, followed by Ca²⁺-mediated ionic crosslinking. Rheological analysis showed that ACE incorporation increased both the storage modulus (G′) and loss modulus (G″) of the hydrogels. Cell viability and morphology were evaluated using Live/Dead staining on day 7, and cellular growth was assessed using an MTT assay on days 1, 4, and 7. Live/Dead staining showed high cell viability in all groups, with few dead cells observed. STO cells on pure alginate hydrogels remained mostly spherical, whereas those on ACE/ALG composite hydrogels exhibited enhanced spreading and formed spindle-shaped cellular networks. Quantitative analysis showed that ACE incorporation significantly increased cell viability compared with the pure alginate group (1ALG, 90.1 ± 1.8%), with the 0.25ACE/2ALG and 0.5ACE/2ALG groups showing the highest viability (P < 0.05). MTT analysis showed that the 0.5ACE/2ALG group exhibited significantly higher metabolic activity than the 1ALG group on day 4. The highest metabolic activity was observed on day 7 (P < 0.05). These results suggest that ACE improves the cytocompatibility and cell-supportive properties of ALG hydrogels, with 0.5ACE/2ALG providing the most favorable formulation for STO cell spreading and growth. Therefore, ACE/ALG composite hydrogels may serve as promising naturally derived and biocompatible scaffold platforms for tissue engineering, with potential application in future cultured meat production.