Comprehensive Analysis of Proteomic Variations Influenced by Culture Conditions in Human Mesenchymal Stromal Cells

Proteomics, a powerful approach allowing the broad analysis of protein expression levels in samples, has been used to characterize mesenchymal stromal cells (MSCs) used in regenerative medicine, focusing on the influence of their tissue of origin, culture media, and passage number. However, the proteomic signatures arising from comprehensive alterations in culture conditions remain to be characterized. Here, we evaluated the effects of major culture parameters, including cell seeding density, detachment methods, media composition, and passage number, on both the cellular and supernatant proteomic profiles of human MSCs. Among the evaluated parameters, the 15-passage condition exerted the most significant impact on the cell samples, with the most significant proteome remodeling, including decreased DNA replication-related proteins and increased lipid metabolism-related proteins. Regarding the supernatant samples, significant differences were found between xeno-free media and serum-containing media, with serum-containing media showing higher secretion of the insulin growth factor-binding protein (IGFBP) family. Further, functional changes included reduction in thrombospondin-1, which is associated with MSC migration. These findings demonstrate that culture conditions significantly influence the characteristics of MSCs used in regenerative medicine, and that a wide proteomics evaluation of both the cells and supernatant is crucial for understanding these characteristics.