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.
This study examined the potential benefits of male specific-pathogen-free (SPF) White Leghorn embryos in cellular agriculture for sustainable and ethical poultry meat production—addressing traditional farming challenges, including disease outbreaks of Salmonella and Avian influenza. We isolated myogenic precursor cells (MPCs) from the thigh muscles (Musculus femoris) of 12.5-day-old embryos from 10 SPF White Leghorns that tested negative for Salmonella. We randomly selected MPCs from three males and three females, isolated them using a modified pre-plating (pp) method, and compared their in vitro development. After 1 h (pp1) and 2 h (pp2) of incubation, they were transferred to a new dish to remove fast-adhering cells and cultured (pp3). Isolated MPCs had a 69% positive reaction to Pax7. During proliferation, no differences were observed in PAX7, MYF5, or MYOD expression between the male and female MPCs. However, after five days of differentiation, the expression of late myogenic factors—MYOG and MYF6—significantly increased in all MPCs. Notably, MYOG expression was 1.9 times higher in female than in male MPCs. This impacted MYMK’s expression pattern. Despite this, the myotube fusion index did not differ between the sexes. Muscle cells from male SPF-laying chicken embryos are promising for developing clean animal-cell-derived protein sources via resource recycling.
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