Mesenchymal Stem Cells Lose the Senescent Phenotype Under 3D Cultivation

Abstract

Abstract Background Three-dimensional (3D) cell culture is widely used in various fields of cell biology. In comparison to conventional two-dimensional (2D) cell culture, 3D cell culture facilitates a more accurate replication of the in vivo microenvironment, which is essential for obtaining more relevant results. The application of 3D cell culture techniques in regenerative medicine, particularly in mesenchymal stem cell (MSC)-based research, has been extensively studied. Many of these studies focus on the enhanced paracrine activity of MSCs cultured in 3D environments. However, few focus on the cellular processes that occur during 3D cultivation. Methods In this study, we studied the changes occurring within 3D-cultured MSCs (3D-MSCs). Specifically, we examined the expression of numerous senescent-associated markers, the actin cytoskeleton structure, the architecture of the Golgi apparatus and the localization of mTOR, one of the main positive regulators of replicative senescence. In addition, we assessed whether the selective elimination of senescent cells occurs upon 3D culturing by using cell sorting based on autofluorescence. Results Our findings indicate that 3D-MSCs were able to overcome replicative senescence. We discovered changes in actin cytoskeleton structure, Golgi apparatus architecture and revealed that 3D cultivation leads to the nuclear localization of mTOR, resulting in a decrease in its active cytoplasmic form. Furthermore, we confirmed that 3D cell culture facilitates the rejuvenation of senescent cells rather than their elimination from the bulk population. Conclusion These novel insights into the biology of 3D-MSCs can be applied to research in regenerative medicine to overcome replicative senescence and MSC heterogeneity as they often pose significant concerns regarding safety and effectiveness for therapeutic purposes.