Optimizing in vitro culture conditions for broiler chicken Pectoralis major muscle satellite cells
Type of DegreeMaster's Thesis
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There is growing interest in understanding how muscle-specific stem cells, or satellite cells (SC), contribute to post-hatch skeletal muscle growth of broiler chickens and their potential role in the development of breast myopathies. It has been established that the development of the Wooden Breast (WB) myopathy is associated with hypoxia, oxidative stress, excessive collagen deposition, and the infiltration of inflammatory cells; all of which are known to alter SC behavior. Functional differences among SC populations can be examined using in vitro analysis. However, cell culture conditions for SC from modern broilers are not standardized in the existing literature. The objective was to optimize in vitro culture conditions for primary SC isolated from the Pectoralis major (PM) of broiler chickens. Two experiments were conducted using primary SC isolated from the PM of Ross 708 × Yield Plus male broilers. Experiment 1 evaluated the impact of initial plating densities of either 10,000, 20,000, 30,000, or 40,000 live cells per 1.8-cm2 culture well. Cell viability was improved with 20,000 or more cells per well (P < 0.0001). Proliferation was similar among densities (P = 0.6965). The expression of myogenic regulatory factors (MRF) indicated that wells plated with 40,000 cells reached confluency by 96 h, and densities at or above 30,000 cells per well improved SC progression through myogenesis (P = 0.0758). Experiment 2 compared a standard culture temperature of 38 °C with broiler PM peripheral muscle temperature of 41 °C to incubate cells. Proliferation was not altered by temperature (P ≥ 0.1148), but MRF heterogeneity of all measured populations was impacted by 96 h (P < 0.0001), indicating a more rapid progression through myogenesis at 41 °C. Apoptosis was increased at 41 °C (P ≤ 0.0302). Finally, the fusion of cells into myotubes was diminished at 41°C (P < 0.0001), but the density of fused cells was similar (P = 0.7551). Myotube characteristics were also altered by culture temperature (P ≤ 0.0806). These observations raise questions about how temperature may provoke primitive, embryonic behavior of SC isolated from post-hatch broilers and indicate that optimal culture temperature may depend on the objective of the experiment. Together, these results help establish a standard protocol for the culture of primary SC from the PM of modern broilers and develop a basis for future functional analysis of SC populations to better understand cellular mechanisms underlying WB development.