Comparative Metabolomic and Transcriptomic Analysis of Two Dimensional and Three Dimensional Mesenchymal Stem Cell Cultures for Improved Therapeutic Applications

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine due to their multipotency and immunomodulatory capabilities. Compared to conventional two-dimensional (2D) monolayer cultures, three-dimensional (3D) spheroid cultures more closely recapitulate the in vivo microenvironment, influencing MSC metabolic activity and therapeutic efficacy. This study utilizes metabolomics and transcriptomics to assess and compare the metabolic activity of MSCs cultured under 2D and 3D conditions, to evaluate how these culture environments affect MSC behavior, proliferation, and function. Our findings indicate that MSCs in 3D spheroids exhibit reduced proliferation, enhanced stemness, and distinct metabolic adaptations, including increased glycolysis and alterations in nutrient metabolism. These changes promote a more quiescent state, which is favorable for tissue repair and immune modulation. Understanding these metabolic adaptations can provide valuable insights for optimizing culture conditions, improving MSCs-based therapies, and identifying novel therapeutic targets and biomarkers. Overall design: Total RNAs were extracted from cell samples of 2D MSCs and 3D spheroids collected at two time points-0 hour (0h) and 24 hours (24h) after culture, to identify differential gene expression between 2D and 3D MSCs.