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Abstract:

To develop an in vitro system to simultaneously assess transport through the human nasal epithelial cells (HNEC) and the effect on ciliary beat frequency (CBF). Cells were grown in ThinCertTM tissue culture inserts in 12-well Cellstar® cell culture plates. The membrane consisted of transparent polyethylene terephthalate (3µm pores) and was coated with collagen. The cell number was determined using a Bürker chamber and 1-1.3*106 cells were plated in each ThinCertTM. Cell viability was assessed by trypan blue exclusion. Five days after plating, HNEC formed microscopically confluent layers. Transport experiments were performed on day 10 or 11. Transport was assessed for 3 compounds: atenolol (paracellular transport), talinolol (P-glycoprotein substrate) and propranolol (transcellular transport). Concentrations were determined by HPLC (fluorescence detection). Data for CBF calculation were collected using an inverted microscope coupled with a high-speed digital camera; 1054 images were captured during 2 seconds. CBF values were determined for each pixel site separately by spectral analysis of the variation of the pixel intensity over time. Fast Fourier Transformation (FFT) was applied to the intensity signal and CBF was computed as the frequency corresponding to the maximal FFT amplitude value in the range from 0 to 20Hz. Viability of cells obtained by enzymatic dissociation from nasal biopsies ranged from 91.8% to 97.1%. Transepithelial electrical resistance (TEER) increased as a function of time in culture and reached a plateau level after 7-9 days. Transport inserts with TEER values over the cell layer below 600 &[Omega].cm2 were excluded from transport experiments. Absorptive apparent permeability coefficient (Papp) values amounted to 0.3±0.2, 0.6±0.2 and 16±5x10-6 cm/s for atenolol, talinolol and propranolol, respectively, illustrating the ability of the system to discriminate between compounds with different transport properties. No statistically significant differences in CBF could be observed when measuring CBF at defined days in culture. Inclusion of chlorocresol (0.01%) decreased CBF (to 55%), but did not alter transport. For the first time, a system was developed in which the effect of compounds on CBF could be followed simultaneously with transport through the HNEC layer. This system could become an interesting tool to study drug candidates for nasal administration.