Purpose: To elucidate the apical and basolateral components of the total unstirred water layer in regular permeability experiment.
Methods: A novel stirring apparatus was constructed to remove the basolateral unstirred water layer. Caco-2 cells were used as the permeability barrier both in Transwell-type and side-by-side apparatuses. Permeability experiments were done with several ionisable compounds at various pH and stirring conditions. The permeabilities of the cell monolayer, the unstirred water layer and the polycarbonate filter were calculated either from experimental data or theoretically.
Results: The unstirred water layer was thicker in the Transwell apparatus than in the side-by-side chamber even in the presence of vigorous basolateral magnetic stirring. Calculations indicated that the apical unstirred water layer is thicker than the basolateral layer. Different cellular permeability coefficients were obtained from the two permeability apparatuses.
Conclusions: An orbital shaker does not produce symmetric hydrodynamics in both chambers of Transwell apparatus. The asymmetric unstirred water layer may complicate the exact analysis of polarized transport.