Transcellular transport and the accumulation of [14C]tetraethylammonium, a typical organic cation, by LLC-PK1 cell monolayers grown on microporous membrane filters were studied. Tetraethylammonium was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. The transcellular transport of tetraethylammonium was saturable, temperature dependent, and sensitive to the pH of the apical side of the monolayers. The apparent Michaelis constant and maximum velocity values for the transport were 67 microM and 222 pmol.mg protein-1.min-1, respectively. Unlabeled tetraethylammonium, amiloride, procainamide, cimetidine, and choline inhibited the basolateral uptake and transcellular transport of [14C]tetraethylammonium. The development of tetraethylammonium transport activity was observed in the differentiating cells. A sulfhydryl reagent inhibited the tetraethylammonium transport at both the basolateral and apical membranes of the LLC-PK1 cells. These findings suggest that these monolayers possess unidirectional transport systems for organic cations, corresponding to the secretion in the renal proximal tubules.

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Dental pulp stem cells (DPSCs) are a good source for tissue regeneration, however, the number of DPSCs in the pulp tissue is limited. Cell propagation is essential for tissue engineering using DPSCs and the cell culture conditions may affect the properties of DPSCs. The purpose of this study was to analyze the effect of cell culture condition, especially dense culture condition, on the property and differentiation pathway of DPSCs. We cultured DPSCs under sparse (sDPSCs; 5 × 10 3 cells/cm 2 ) or dense (dDPSCs; 1 × 10 5 cells/cm 2 ) conditions for 4 days and compared their properties. The populations of CD73 + and CD105 + cells were significantly decreased in dDPSCs. Both groups showed multi-differentiation potential, but mineralized nodule formation was enhanced in dDPSCs. The phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) proteins was promoted in dDPSCs, and alkaline phosphatase (ALP) mRNA expression in dDPSCs was abolished in the presence of pan-PI3K and FAK inhibitors. dDPSCs implanted into mouse bone cavities induced more mineralized tissue formation than sDPSCs and control. These findings indicate that dense culture conditions modified the properties of DPSCs and gave rise to osteogenic-lineage commitment via integrin signaling and suggest that dense culture conditions favor the propagation of DPSCs to be used for mineralized tissue regeneration.

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