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Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively . All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection., Modeling intestinal infection with NASA biotechnology: A new 3-D intestinal co-culture model with macrophages to study enteric infection Using spaceflight analog bioreactor technology, Cheryl Nickerson at Arizona State University and collaborators developed and validated a new three-dimensional (3-D) intestinal co-culture model containing multiple differentiated epithelial cell types and phagocytic macrophages with antibacterial function to study infection by multiple pathovars of Salmonella. This study is the first to show that these pathovars (known to possess different host adaptations, antibiotic resistance profiles and disease phenotypes), display markedly different colonization and intracellular co-localization patterns using this physiologically relevant new 3-D intestinal co-culture model. This advanced model, that integrates a key immune cell type important for Salmonella infection, offers a powerful new tool in understanding enteric pathogenesis and may lead to unexpected pathogenesis mechanisms and therapeutic targets that have been previously unobserved or unappreciated using other intestinal cell culture models.