INTRODUCTION: Mesenchymal Stem Cells (MSC) possess ability to release cytokines and growth factors that suppress immune responses and stimulate tissue regeneration. Wharton’s Jelly-derived MSC (WJ-MSC) in the addition to the strong adjuvant properties are characterized by low immunogenicity. AIM(S): The aim of this study is to verify immunomodulatory properties of WJ‑MSC after TNFα and IFNγ stimulation in vitro, by comparative analysis of the expression of cytokines and growth factors they produce. METHOD(S): WJ-MSC isolated from human umbilical cords were cultured in closed system that provides a constant 5% oxygen concentration. We compared immunomodulatory properties of WJ-MSC in 2D or 3D structures (scaffolds) made in our laboratory. Both of those cell populations were cultured in medium with/ without stimulate factors: TNF‑α and IFN‑γ. After stimulation, 2D and 3D cell cultures were characterized with quantitative RT-PCR for the expression of various cytokines and growth factors with non-stimulated 2D cells as an internal control. WJ-MSC grown in 3D were also characterized by live/dead cells presence which were labeled with calcein AM/ethidium homodimer. RESULTS: The obtained results indicated increased expression of mRNA in 3 D structures vs. control 2 D cells for almost all analyzed cytokines: IL‑6, TGF‑β1 , BDNF, GDNF, EGF, bFGF. Moreover, TNF‑α and IFN‑γ stimulation causes even further increase of mRNA expression of those cytokines in 3 D cultures compared to non-stimulated 2 D control. In our scaffolds models, the intercellular connections which were labeled in live cells with calcein AM have been observed already after 24h of culture and are visible also during the next days of analysis. CONCLUSIONS: Finally we can conclude, that WJ-MSC produce immunomodulatory factors, and their expression can be modulated by stimulation with chosen cytokines and 3D microenvironment. Such properties of WJ-MSC are important for the potential therapeutic application in the treatment of the diseases of inflammatory and autoimmune origin. FINANCIAL SUPPORT: The work was supported by National Centre for Research and Development grant No STRATEGMED1/234261/2/NCBR/2014.