Culture of human nasal polyp epithelial cells at an air-liquid surface

Abstract

To study the human nasal ciliated epithelial cells at an air-liquid surface (ALI) so as to establish a reliable cell culture model for nasal mucociliary transport study. The human nasal ciliated epithelial cells were cultured by low-temperature enzymatic digestion method at an air-liquid surface, the cell growth behavior was observed under the inverted phase-contrast microscope, the proliferation, confluence and differentiation of cultured cells were examined by scanning electron microscope and immunocytochemistry, the basal and stimulated ciliary beat frequency (CBF) of cultured epithelial cells were measured by using high-speed digital microscopic imaging system. Prism 4.0 software was used to analyze the data. (1) Under microscope, the cells on transwell membrane adhered well at 24 h and locked tightly to display with a cobblestone-like appearance; the monolayer cells got confluence to 80%-90% after one-week submersion culture, and thereafter exposed to air-liquid interface. (2) Under scanning electron microscope, the cilia and also the small microvilli could be observed to protrude from the cell's surface at ALI day 7; the ciliated cells differentiated well and distributed in cluster; goblet cells and nonciliated columnar cells distributed between ciliated cells. (3) Immunocytochemistry of β-tubulin IV and zona occludens-1 showed a good confluence and differentiation of cilia in cultured epithelial cells at ALI day 14, and the percentage of ciliated epithelial cells was 50%-60 %. (4) The basal CBF of cultured epithelial cells was (8.42 ± 1.24), (8.71 ± 1.11), (9.17 ± 1.11), (8.89 ± 0.91), (8.99 ± 0.91) Hz at ALI day 7, 14 , 21, 28, 35, respectively, no significant difference was found among them(F = 1.451, P > 0.05). (5) At the concentration of 100 µmol/L ATP, an exogenous stimulating agent, significantly increased the CBF of cultured epithelial cells. Air-liquid interface cultured human nasal epithelial cells by enzymatic digestion method manifest with good confluence and differentiation status; the cells could maintain differentiated morphology and physiological function close to in vivo epithelium for a long term, therefore, it may serve as an ideal cell model for mucociliary transport study.