Establish a cultured meat model with adjustable muscle-to-fat ratios based on muscle satellite cells and fibroadipogenic progenitor cells in an optimized gelatin-mTG scaffold.

Cell-cultured meat offers a sustainable alternative to conventional meat; however, existing monocellular models fall short in replicating its texture and sensory qualities. This study developed an economical gelatin/microbial transglutaminase scaffold to construct a multicellular beef analogue. Muscle satellite cells (MuSCs) and Fibroadipogenic progenitor cells (FAPs) were isolated by flow cytometry. Under 2D culture, MuSCs exhibited enhanced myogenesis (MYH3, MyoD, MyoG upregulated 1.5-2.4-fold), while FAPs showed adipogenic potential (ADIPOQ, FABP4 elevated). Scaffold variants were optimized: GOS (Gelatin-Oriented Scaffold) (muscle-supportive) and GAS (Gelatin-Sodium Alginate Scaffold) (fat-supportive), both supporting high proliferation. Transition to 3D culture further promoted differentiation, increasing myogenic/adipogenic gene expression (2-2.3-fold, p < 0.01) and extracellular matrix secretion (∼2-fold). After 14 days, constructs displayed uniform pink coloration. Post-frying, color parameters (a* [redness], L* [lightness]) were comparable to conventional beef (p > 0.05). This scalable approach creates structured, sensory-comparable cultured meat.