Investigation of scaffold manufacturing conditions for 3-dimensional culture of myogenic cell line derived from black sea bream (Acanthopagrus schlegelii).

Culturing fish myogenic cells in vitro holds significant potential to revolutionize aquaculture practices and support sustainable food production. However, advancement in in vitro culture technologies for skeletal muscle-derived myogenic cells have predominantly focused on mammals, with limited studies on fish. Scaffold-based three-dimensional (3D) culture systems for fish myogenic cells remain underexplored, highlighting a critical research gap compared to mammalian systems. This study evaluated the effects of scaffold composition and manufacturing methods on cellular growth in the 3D culture of black sea bream (Acanthopagrus schlegelii) myogenic cells. Scaffolds were manufactured using three natural polymers: black sea bream-derived extracellular matrix (ECM), sodium alginate, and gelatin. Two scaffold types were tested: "cell-laden scaffolds" prepared by mixing cells into the pre-scaffold solution followed by gelation, and "cell-seeding scaffolds" produced by freezing, gelation, and lyophilization before cell inoculation. Scaffold characteristics, including pore size, porosity, swelling ratio, and degradation rate, were assessed. Cell-seeding scaffolds exhibited relatively larger pore size, higher porosity, and higher degradation rate, while cell-laden scaffolds had higher swelling ratios. When black sea bream myogenic cells were cultured in these scaffolds, cell-seeding scaffolds supported cellular growth, particularly when composed of 3% sodium alginate and 4% gelatin with any concentration of ECM. In contrast, cell-laden scaffolds did not support cellular growth regardless of their composition. These findings provide fundamental insights for optimizing scaffold properties to develop more optimized conditions for 3D culture of fish muscle lineage cells.