biostudies-literatureUnknown3Barrila JThree-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 <i>Salmonella</i> 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 <i>Salmonella</i>, 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 <i>Salmonella</i> pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). ??In addition, <i>Salmonella</i> were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively?. All <i>Salmonella</i> 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.NPJ microgravity10https://www.ebi.ac.uk/biostudies/studies/S-EPMC5460263biostudies-literatureThree-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study <i>Salmonella enterica</i> colonization patterns.PMC5460263Yang JClemett SJRoland KLNickerson CACrucian BELiu YNelman-Gonzalez MAQuiriarte HForsyth RJBrenneman KBarrila JNydam SDDavis RRCrabbe ASams CSarker SFOtt CMLoscher CfalseThree-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study <i>Salmonella enterica</i> colonization patterns.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 <i>Salmonella</i> 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 <i>Salmonella</i>, 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 <i>Salmonella</i> pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). ??In addition, <i>Salmonella</i> were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively?. All <i>Salmonella</i> 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.2017-01-01T00:00:00Z20172021-02-20T21:31:19Z2019-03-27T02:46:58ZS-EPMC54602632864963210.1038/s41526-017-0011-2