关键词: 成肌细胞；细胞培育肉；微载体；转瓶；优化培养工艺

Abstract: With the rapid development of the cultivated meat industry, large-scale cell culture technology applied to cultivated meat production has received extensive attention. In this study, two microcarriers, Cytodex 1 and 3D TableTrix™, were used for suspension culture of myoblasts in spinner flasks to select a more suitable one for large-scale culture of myoblasts, and then the effects of different culture conditions on the large-scale culture of cells were explored. The microscopic results showed that Cytodex 1 was spheres with a smooth surface and no macroporous structure, and 3D TableTrix™ was irregular spheres with a porous surface and large pores. The results of cell culture in spinner flasks showed that the cells grew on 3D TableTrix™ more efficiently, and after 10 days of culture, the cell yield was 8.97 × 105 cells/mL. The optimal conditions for cell attachment were intermittent stirring at 40 r/min for 10 min with intervals of 50 min. The best cell culture efficiency was obtained by using cell inoculation density of 1 × 105 cells/mL, microcarrier density of 2 mg/mL, agitation speed of 40 r/min, and initial culture serum concentration of 20% as well as replacing the medium with a new one containing 10% serum (V/V) after 24 h of culture, and replacing 50% of the medium with a new one with every other day. By bead to bead transfer of the microcarriers, the cells were transferred to a 2 L spinner flask. Finally, 1.07 × 109 cells were harvested with a survival rate of more than 95%. Microcarriers loaded with cells could be cryopreserved and resuscitated, and no significant differences in proliferation status and morphology were observed compared with thawed cells. The optimized microcarrier based culture process will have promising applications in the industrial scale production of cultivated meat.

Key words: myoblast; cultivated meat; microcarriers; spinner flask; optimized culture system