Introduction: Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies.To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids.Methods: Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor.Lymphocytes were pre-incubated with Con A (ConA) and then co-cultured with MSC in the matrix with addnl. ConA in the perfusing medium.Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run.Paraffin sections of bioreactor matrixes were analyzed by proliferating cell nuclear antigen (PCNA)-specific staining to determine MSC proliferation.Immune modulatory capacity was defined by daily anal. of cytokine secretion profiles (TNFα, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70s GM-CSF).Results: Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFα (96.8 ± 4.8%) and IFNγ (88.7 ± 12.0%) in 3D co-cultures.In contrast, other cytokines (IL-1, IL-6, IL-12) were induced.Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only.Conclusions: In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.