An efficient and economical way to obtain porcine muscle stem cells for cultured meat production

Cultured meat technology is an emerging and promising strategy for animal protein production. Muscle stem cells are regarded as important seed cells for generating muscle fiber in vitro because of their proliferative and myogenic differentiation potential. However, current approaches for the isolation and purification of muscle stem cells are low-yield and high-cost, limiting the industrial production of cultured meat. Here, we reported an efficient and economical protease combination consisting of pronase and dispase II for the isolation of primary muscle stem cells, achieving 5.06 ± 0.12 × 10⁶ nucleated cells and 3.19 ± 0.19 × 10⁶ Pax7⁺ cells from 1 g of porcine muscle tissue. Furthermore, by investigating the effect of initial purity on the proliferation and differentiation potential of muscle stem cells, we found that higher purity of initial muscle stem cells promoted the maintenance of myogenic properties of cells after expansion but reduced the total number of obtained cells. Based on nucleated cells isolated from 1 g of porcine muscle, muscle stem cells purified by 0.5 h of pre-plating yielded 2.19 ± 0.16 × 10⁸ cells with myogenic differentiation capacity after 20 days of expansion, which was 5-fold higher than those purified by fluorescence-activated cell sorting (FACS). Therefore, a modified approach was developed to obtain porcine muscle stem cells for cultured meat production, involving tissue digestion with the pronase and dispase II combination and purification through pre-plating for 0.5 h. This approach was simple, efficient, and economic, which would facilitate the industrial production of cultured meat.