The tight junction formation in Caco-2 cell monolayers was compared after 9 and 13 d of culture. Four different sized paracellular markers were simultaneously applied to the apical side of the monolayers. The transepithelial resistance and permeability coefficient of mannitol for the monolayers cultured for 13 d were 17.4 and 0.095 times those after 9 d, respectively. The tight junction structure developed during that period. The pore permeation model involving two different sizes was used to quantitatively evaluate the paracellular pathways. The results suggest that there were two-different sized (large and small) pathways in the monolayers cultured for 13 d, while there was only a single large pathway in those cultured for 9 d. The small pathway in the monolayers cultured for 13 d might be a major permeation pathway for the paracellular permeation of urea and the equivalent cylindrical pore radius of the small pathway was less than 0.4 nm, suggesting development of the tight junctions after 13 d.
