Isolation of high-purity muscle stem cells through ice-cold treatment method for the production of cell cultured meat.

Cell cultured meat (CCM) is regarded as a viable alternative in the future meat market due to its traceable origins and environmental friendliness. The production of CCM relies primarily on the efficient in vitro expansion of pure muscle stem cells (MuSCs). However, conventional methodologies for isolating MuSCs are often associated with technical complexities, high costs, low cellular purity, and poor stemness of the seed cells. Therefore, this study aimed to use the ice-cold treatment (ICT) method to isolate and purify porcine MuSCs and further characterize their expansion characteristics in vitro. The results indicated that cells harvested from unpurified muscle-derived cells treated at around 0 °C for different durations could be cultured in vitro for an extended period, while maintaining their differentiation potential. Among them, cells harvested after 30 min of ICT exhibited a high proliferation rate and excellent activity. Furthermore, the MuSCs obtained through this method can be harvested in large quantities through three-dimensional large-scale cultivation in a bioreactor. The harvested cells can subsequently be used to produce meatballs with a texture resembling that of real meat. This method can reduce the cost of obtaining seed cells and accelerate the research on CCM production, thereby providing further inspiration for the industrialized production of CCM.