Untargeted metabolomics reveals metabolic reprogramming and key metabolites in adherent and suspension Vero cells.

INTRODUCTION: Vero cells are the primary host for industrial vaccine production, and high-density suspension culture is the predominant manufacturing approach. However, this method faces major challenges, including low shear tolerance, difficulty adapting to serum-free conditions, metabolic imbalance, prolonged acclimation periods, and batch-to-batch variability. Furthermore, the metabolic reprogramming mechanisms underlying suspension adaptation remain poorly understood. METHODS: We established serum-free suspension adaptation of adherent Vero cells using reduction serum levels strategy. Biological characteristics of suspension-adapted cells were analyzed through morphological observation, cell viability assays, and karyotyping. A recombinant rabies vesicular stomatitis virus infection model was used to assess viral susceptibility and replication capacity. Untargeted liquid chromatography-tandem mass spectrometry metabolomics was then utilized to systematically characterize intracellular metabolic profiles of Vero cells cultured under adherent and suspension conditions. Differential metabolites were identified, and key regulatory pathways were evaluated to elucidate the metabolic mechanisms of suspension adaptation and identify potential optimization targets. RESULTS: Suspension-adapted Vero cells exhibited uniform morphology, stable genetic characteristics, and robust proliferative capacity, without evidence of degeneration or mutation. Viral sensitivity assays demonstrated efficient viral adsorption and replication, yielding stable viral titers and confirming suitability for vaccine production. Metabolomic analysis identified 119 metabolites (32 upregulated, 47 downregulated, and 40 unchanged), with significant enrichment in alanine/aspartate/glutamate metabolism, glycine/serine/threonine metabolism, and the tricarboxylic acid cycle. Suspension cells displayed Warburg-like metabolic features, including enhanced glycolysis, increased lactate accumulation, and pronounced upregulation of succinate and malate. L-serine and ethanolamine phosphate were strongly upregulated, whereas polyunsaturated fatty acids were significantly reduced. Suspension adaptation involved coordinated remodeling of energy, amino acid, nucleotide, and lipid metabolism; enhancing shear tolerance through membrane lipid remodeling; improving antioxidant capacity via serine metabolism; and supporting high-density proliferation through glycolytic reprogramming. DISCUSSION: This study established a serum-free suspension adaptation system for Vero cells with stable phenotypic characteristics and high viral susceptibility while elucidating metabolic regulatory mechanisms that underlie suspension adaptation. The findings provide metabolic targets and a theoretical foundation for rational medium design, targeted supplementation strategies, and development of genetically engineered cell lines with enhanced shear resistance.