Effect of Serum and Oxygen on the In Vitro Culture of Hanwoo Korean Native Cattle-Derived Skeletal Myogenic Cells Used in Cellular Agriculture

Abstract

Skeletal muscle-derived myogenic cells (SKMCs) are novel protein sources capable of replacing animal meat. However, SKMCs have not been commercialized owing to poor productivity and the high cost of in vitro cell culture. Therefore, we cultured SKMCs in varying serum (5-20%) and oxygen concentrations (5-20%) to investigate the parameters that most impact cell productivity (serum, hypoxia, and culture medium) and examined cell proliferation ability and genes involved in myogenesis/proliferation/apoptosis/reactive oxygen species (ROS). In fetal bovine serum (FBS) groups, hypoxia induction doubled cell number, and the 20% FBS/normoxia group exhibited similar cell numbers as 5% FBS/5% hypoxia, confirming that 5% hypoxia reduced serum requirement by four-fold. The use of 20% FBS downregulated MTF5/MYOD1/MYOG/MYH1, whereas hypoxia induction with ≤10% FBS upregulated them. Although 20% FBS lowered TERT expression through rapid cell proliferation, NOX1, a major factor of ROS, was suppressed. DMEM/F12 demonstrated better differentiation potential than F10 by upregulating MYF3/MYOD1/MYOG/MYH1 and downregulating MSTN, particularly DMEM/F12 with 2% FBS/5% hypoxia. The myogenic fusion index was higher in DMEM/F12 without FBS than in DMEM/F12 with FBS (0.5-5%); however, the total nuclei number was reduced owing to apoptosis. Therefore, high serum levels are essential in influencing SKMC growth, followed by hypoxia as a synergistic component.