title: Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.
abstract: The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.
authors.affiliation: Faculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine.
authors.forename: Neveda
authors.initials: N
authors.lastname: Naz
authors.forename: Dominic C
authors.initials: DC
authors.lastname: Mills
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.forename: Nick
authors.initials: N
authors.lastname: Dorrell
mesh_terms: D001431:Bacteriological Techniques / Q000295:instrumentation / Q000379:methods*; D018938:Caco-2 Cells; D016123:Campylobacter jejuni / Q000502:physiology*; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D015194:Diffusion Chambers, Culture; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology*