Comparative Study of Redox Status of MDCK Cells in Chicken Embryo Extract Versus Fetal Bovine Serum

Fetal bovine serum (FBS) is the standard supplement for cell culture, yet we previously demonstrated that it drives hyper-proliferation and phenotypic drift in Madin-Darby canine kidney (MDCK) cells, compromising their epithelial identity and ciliogenesis. In contrast, a modified chicken embryo extract (CEE) preserved these intrinsic properties, maintaining a stable and physiologically relevant phenotype. To elucidate the metabolic mechanisms driving these distinct cellular fates, we performed a comparative analysis of redox status and metabolomic profiles. We found that FBS forces a metabolic shift toward oxidative phosphorylation, resulting in mitochondrial stress characterized by elevated mitochondrial reactive oxygen species (mtROS), calcium overload, and the accumulation of uremic toxins like hippuric acid. Conversely, CEE established a balanced redox environment. Although CEE induced higher intracellular signaling ROS via NADPH oxidase 1/2, it prevented oxidative damage by upregulating antioxidant transcription factors, such as nuclear factor erythroid 2-related factor 2, and enzymes such as Mn superoxide dismutase. Additionally, metabolomic analysis revealed that CEE is enriched with antioxidants (ascorbic acid, proline) and signaling molecules (5-hydroxyindole-3-acetic acid). These findings indicate that while FBS imposes a metabolic burden leading to cellular stress, CEE provides a favorable metabolic microenvironment that supports homeostasis and epithelial integrity, validating its superiority as a culture supplement.