Production of cultivated meat with stably proliferated porcine muscle stem cells and edible scaffolds

• VPA increased the proportion of Pax7 + /MyoD + cells during porcine MuSC expansion. • VPA treatment significantly maintained the myogenic potential of porcine MuSCs. • A VPA/cytokine combination enabled porcine MuSCs to proliferate for 60 days. • The edible SF-SA scaffolds combine low cost, high strength, and biocompatibility. To achieve large-scale production of high-quality cultivated meat, it is crucial to develop a culture system that efficiently expands muscle stem cells (MuSCs) while maintaining their differentiation potential, as well as an edible scaffolding system that combines low cost, good mechanical strength, and high cell compatibility. In this work, we identified that valproic acid (VPA) was able to promote the proliferation of porcine MuSCs while significantly maintaining their myogenic differentiation potential. Mechanistically, VPA significantly maintained the population of Pax7 + /MyoD + cells through the activation of Notch signaling. Moreover, an efficient expansion strategy for porcine MuSCs was developed through the combined application of VPA and four cytokines, which enabled long-term stable proliferation of porcine MuSCs for 60 days and remained capable of generating multinucleated myotubes. Furthermore, we developed an edible scaffold composed of silk fibroin (SF)-sodium alginate (SA) composite hydrogels by 3D printing and manufactured cell-based pork with outstanding texture by efficient myogenesis of porcine MuSCs on the SF-SA scaffold. This study provides a feasible strategy for the large-scale and stable expansion of MuSCs and the fabrication of structured cultivated meat tissues.