Immunomodulatory Properties of Cytokine-preconditioned Compact-bone Derived Mesenchymal Stem Cells Cultured in 2D and 3D Culture Conditions

Abstract

Abstract. 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-α and IFN-γ) 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-α and IFN-γ alone and in combination for 24 hours. The levels of immunomodulatory factors (PGE2, IL-6, IL-10, TNF-α, IFN-γ) were measured by ELISA. The immunomodulatory properties of MSCs were examined by macrophage inflammation and lymphocyte co-culture assays. Our results showed that TNF-α and IFN-γ more effectively increased the secretion of PGE2 and IL-6 by 3D spheroid MSCs compared with 2D MSCs culture.Сytokine-preconditioned 3D spheroid MSCs significantly decreased the production of TNF-α, IFN-γ, IL-10 in splenic lymphocytes and TNF-α and IL-6 in macrophages. Сytokine-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-α suppressed more effectively in vitro immune response than preconditioning with IFN-γ 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.

TNF-α Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in an Experimental Model of Atherosclerosis

“…New approaches and strategies to improve the immunomodulatory and regenerative capacities of MSCs in treating AS should be further investigated. One such strategy is preconditioning under ex vivo conditions in a defined environment using various biological, chemical, or physical factors [ 13 , 26 , 27 , 28 ]. To improve the therapeutic efficacy of MSCs, variable preconditioning approaches have been suggested, including but not limited to cytokines, hypoxia, a 3D culture, the genetic modification of MSCs, and autophagy modeling [ 13 , 26 , 27 ].…”