Immunomodulatory properties of cytokine-preconditioned compact-bone derived mesenchymal stem cells cultured in 2D and 3D culture conditions

Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties and therefore represent a promising therapeutic tool for the treatment of immune-related diseases. Recently, preconditioning has been proposed as a strategy to improve the immunomodulatory activity of MSCs. In this study, we focused to investigate the effects of preconditioning with pro-inflammatory cytokines (TNF-alpha and IFN-gamma) on immunomodulatory activity of mouse compact bone-derived MSCs (CB MSCs) in 2D and 3D culture conditions. Mouse CB MSCs for the 2D condition were cultured in a standard tissue culture plate. 3D spheroid MSCs were formed by enforced composing in Nunclon Sphera 96-well U-bottom plate. MSCs in 2D and 3D spheroid cultures were preconditioned with TNF-alpha and IFN-gamma alone and in combination for 24 hours. The levels of immunomodulatory factors (PGE2, IL-6, IL-10, TNF-alpha, IFN-gamma) were measured by ELISA. The immunomodulatory properties of MSCs were examined by macrophage inflammation and lymphocyte co-culture assays. Our results showed that TNF-alpha and IFN-gamma more effectively increased the secretion of PGE2 and IL-6 by 3D spheroid MSCs compared with 2D MSCs culture. Cytokine-preconditioned 3D spheroid MSCs significantly decreased the production of TNF-alpha, IFN-gamma, IL-10 in splenic lymphocytes and TNF-alpha and IL-6 in macrophages. Cytokine-preconditioned 3D spheroid MSCs increased IL-6 and IL-10 levels in lymphocytes and macrophages respectively. It was found that 3D spheroid MSCs more effectively suppressed the proliferation of T cells compared with 2D MSCs culture. Moreover, preconditioning of 3D spheroid MSCs using TNF-alpha suppressed more effectively in vitro immune response than preconditioning with IFN-gamma or with the combination of two cytokines. Thus, our data suggest that preconditioning with pro-inflammatory cytokines more effectively increases the immunomodulatory activity of 3D MSC spheroids compared to 2D MSC culture.