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Partly because of inherent limitations of in vivo models, the cellular mechanisms underlying the process of bacterial translocation across the intestinal epithelial barrier are incompletely understood. We therefore used the Caco-2 intestinal cell line as an in vitro model to examine the bacterial translocation process under controlled conditions. Caco-2 cells were grown on porous membranes in the upper compartment of a two-compartment system. Caco-2 cells were cultured for 7, 14, 21, or 28 days. Cellular confluence and tight junction integrity were verified by measurements of dextran permeability and transepithelial electrical resistance. Bacterial translocation was measured by culturing the bacteria (E. colt C25) that were able to cross the Caco-2 cell monolayer. The passage of E. coli C-25 and dextran across the Caco-2 monolayer was higher and the transepithelial electrical resistance lower after 7 days of culture than after 14 or 21 days of culture. The Caco-2 cells became impermeable to dextran blue after 14 days of culture with an average transepithelial electrical resistance of 173.1 ± 9.24 ohms · cm2. When increasing doses of (102-109 colony-forming units) of E. coli were tested in 14-day-old Caco-2 monolayers, bacterial translocation occurred in a time- and dose-dependent fashion. Once cellular confluence and tight junction integrity have been established, bacterial translocation across Caco-2 cells appears to be a time- and dose-dependent process.