Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi, Jiangsu 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi, Jiangsu 214122, China; Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, Wuxi 214122, China.
Food Res Int. 2022 Dec;162(Pt B):112206. doi: 10.1016/j.foodres.2022.112206. Epub 2022 Nov 19.
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 × 10 nucleated cells and 3.19 ± 0.19 × 10 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 × 10 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.
培养肉技术是一种新兴的、有前景的动物蛋白生产策略。肌肉干细胞因其具有增殖和肌源性分化潜能，被认为是体外生成肌纤维的重要种子细胞。然而，目前用于分离和纯化肌肉干细胞的方法产量低、成本高，限制了培养肉的工业化生产。在这里，我们报告了一种由胰蛋白酶和中性蛋白酶 II 组成的高效、经济的蛋白酶组合，用于分离原代肌肉干细胞，从 1g 猪肌肉组织中可获得 5.06±0.12×10 个有核细胞和 3.19±0.19×10 个 Pax7 细胞。此外，通过研究初始纯度对肌肉干细胞增殖和分化潜能的影响，我们发现初始肌肉干细胞的纯度越高，细胞扩增后维持其成肌特性的能力越强，但获得的细胞总数越少。基于从 1g 猪肌肉中分离出的有核细胞，通过 0.5h 预铺板对肌肉干细胞进行纯化，可获得 2.19±0.16×10 个具有成肌分化能力的细胞，在经过 20 天的扩增后，这一数量是荧光激活细胞分选（FACS）纯化的 5 倍。因此，我们开发了一种改良的方法来获得用于培养肉生产的猪肌肉干细胞，该方法涉及使用胰蛋白酶和中性蛋白酶 II 混合物进行组织消化，以及通过 0.5h 的预铺板进行纯化。这种方法简单、高效、经济，将有助于培养肉的工业化生产。
