Professional phagocytes such as neutrophils, macrophages, and dendritic cells (DCs) actively engulf microbes. In this study, we developed a three-dimensional (3D) in vitro infection model for investigating the cross-talk between phagocytes and microbes in inflammation. A culture system using a nanofibrous scaffold (NFS) having multi-planar pores in a 3D structure was used for coculture of Staphylococcus aureus (S.aureus) and phagocytes in the same as well as different compartments. Surface-seeded S. aureus and phagocytes were able to adhere to nanofibers inside the NFS and phagocytes migrated to and engulfed S. aureus in a 3D manner. The addition of formyl peptide receptor antagonists decreased the phagocytic rate in 3D NFS but not in two-dimensional (2D) culture dishes. The migration of phagocytes to S. aureus was evaluated in an NFS-based layer-by-layer culture system. Neutrophils, macrophages, and DCs cultured in an upper NFS migrated to the lower NFS containing bacteria. Cytokine and chemokine secretion patterns by neutrophils cultured with S. aureus differed between 2D and 3D culture conditions. DCs migrated to neutrophils phagocytosing bacteria and then engulfed neutrophils in 3D in vitro culture. In addition, neutrophils and macrophages in the upper NFS migrated to bacteria-infected lung epithelial cells in the lower NFS of the layer-by-layer system. S. aureus-infected lung epithelial cells stimulated the secretion of tumor necrosis factor (TNF)-α and IL-1ain 3D culture condition, but not in 2D culture. Therefore, NFS-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo.
