Transport of anti-allergic drugs across the passage cultured human ...

206 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210Fig. 1. Time-course of TEER across passaged human nasal epithelial cellmonolayer during 2 weeks; () mean value of passages 2–4 (n = 9).study (Yoo et al., 2003), transport studies were conductedafter 4–8 days of seeding when the TEER value maintained800–1200 cm 2 .The characteristics of maximum TEER appearing in 2days after seeding was different from that of the rabbittracheal epithelial cell monolayer, primary human alveolarepithelial cells or air-interfaced primary rabbit conjunctivalepithelial culture (Mathias et al., 1995; Elbert et al., 1999;Yang et al., 2000). Relatively high TEER value was alsoreported on the collagen matrix-based human nasal primaryculture using air-liquid interface culture (Agu et al., 2001).High TEER value at the early phase of the culture could bedue to the influence of culture media (DMEM containing 10%FBS), since most of respiratory epithelial cells are culturedin serum-free media, such as PC-1 medium for the primaryculture of rabbit tracheal and conjunctival epithelial cells andDME-F12 for human primary nasal culture (Mathias et al.,1995; Yang et al., 2000; Agu et al., 2001). It could be speculatedthat the serum, an important source of extracellularmatrix (e.g., laminin), promoted cell proliferation, adhesionfactors, and/or anti-trypsin activity (Freshney, 1994).3.2. Morphological studies of passaged human nasalmonolayer cultureIn order to estimate the barrier properties of epithelialcells, morphological studies, bioelectric determination, andnon-electrolyte solute permeability are commonly investigated.In addition to TEER value, SEM, and TEM wereobserved for passage 2 culture on day 5 after seeding. Asshown in Fig. 2(A), tight junctions were clearly observedin TEM (indicated by a white arrowhead). Microvilli andincomplete cilia were also observed at the apical side ofepithelial cells. However, SEM results showed less prominentcilia or denuded ciliated cells of passage 2 in LCCmethod on day 5, which was consistent with the report onrabbit tracheal epithelial cell culture using the same method(Mathias et al., 1995). The epithelial monolayers developed aFig. 2. Morphological appearance of passage 2 human nasal epithelialmonolayer after 5 days of seeding under the transmission and scanning electronmicroscopy. Plate A shows the tight junction (indicated by a white arrow)and the cilia (indicated by a black arrow) (8000× magnification). Plate Bdisplays a cuboidal appearance of passage 2 human nasal epithelial monolayers(600× magnification). The insert shows a SEM of passage 2 humannasal epithelial monolayer exhibiting denuded cilia (4500× magnification).cuboidal shape, as shown in Fig. 2(B), which were observedto be broader and shorter than the “native” pseudo-stratifiedcolumnar epithelial cells. Even though the differentiation ofthe cell monolayers was incomplete, this passaged culturemodel seems to be suitable for drug transport studies sincethe development of the tight junction was completed within2–3 days, which can be advantageous due to reduced experimenttime and cost.3.3. The effect of lipophilicity on the permeability acrossthe human nasal cell monolayerIn preliminary studies, all the model drugs were stable intransport medium and the integrity of the monolayer checkedby the change of TEER was maintained for 60 min of transportstudies (data not shown).