Abstract: 【Objective】 The objective of this study was to explore the differentiation effect of porcine muscle stem cells in three-dimensional hydrogels, and to provide guidance for inducing muscle stem cells to differentiate into muscle tissue in vitro.【Method】 Some porcine muscle stem cells were respectively induced to differentiate under the conditions of 2D and 3D (2D condition means culturing cells in cultured dishes; 3D condition means culturing cells in hydrogels). The RNA and protein samples of porcine muscle stem cells cultured in 2D were collected respectively at proliferation, pre-differentiation, early differentiation, mature differentiation and late differentiation, and those in 3D were also collected respectively at day 7 and day 14 of differentiation. Then, RT-qPCR was used to compare the expression levels of the myogenic-related genes including the genes of MYOG, CAV-3, MyHC-slow, and MyHC-2a, under 2D and 3D differentiation conditions. Correspondingly, Western Blot was used to detect the expression levels of MyHC protein and MYOG protein in the two conditions. Moreover, immunofluorescence staining was used to observe the myotubes formed in cell culture dishes and hydrogels. Further, we analyzed the amino acid content and composition of the cultured muscle tissue by an amino acid automatic analyzer at day 14 of differentiation. 【Result】 The porcine muscle stem cells started to fuse to form myotubes at day 3 of differentiation in 2D. Myotubes formed in 2D matured at day 7 and divorced from culture dish afterwards. The porcine muscle stem cells were still globe and had low expression of MYOG and CAV-3 at day 7 of differentiation in 3D. Multinucleated myotubes formed at day 14 and the expression of MYOG and CAV-3 reached levels of 2D differentiation. The cells in hydrogels had higher expression of terminal differentiation genes MyHC-slow and MyHC-2a than the cells in culture dishes. The expression of MyHC-slow was 12 times that at day 7 in 2D and the expression of MyHC-2a was 4 times that at day7 in 2D, but the expression of MyHC protein was only 1/6 that at day 7 in 2D. Amino acid analysis results showed that the contents of 17 hydrolyzed amino acids in cultured muscle tissue were all lower than those in pork, and the radio of essential amino acids was also lower in cultured muscle tissue, but the radio of flavor amino acids was higher. 【Conclusion】 Porcine muscle stem cells can differentiate into myotubes in 3D collagen hydrogels in vitro, and the 3D conditions are positive to the expression of myogenic differentiation related genes, but further research is needed to achieve high expression of MyHC protein. The flavor amino acid content of the muscle tissue cultured in this way is high, which may mean good flavor.

Key words: porcine muscle stem cells,  , differentiation,  , hydrogels,  , myotubes,  , cultured meat