Ciliary and Secretory Differentiation of Normal Human Middle Ear Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Recent technical advances allow serial culture of normal human middle ear epithelial (NHMEE) cells. However, the ciliary differentiation of these cells has not been achieved. The purpose of this study was to establish a culture system to differentiate serially cultured NHMEE cells into ciliated cells. If the ciliated cells were to develop, the percentage of ciliated cells and secretory cells throughout the duration of culture would be measured. We also examined the levels of mucin and lysozyme secretion and their mRNAs in a time-dependent manner. MATERIALS AND METHOD: Normal appearing human middle ear mucosa was harvested and subcultured after enzymatic disaggregation. These cells were differentiated in air-liquid interface (ALI) culture for 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks after confluence. On each culture period, the ratio of ciliated cells and secretory cells and the amount of mucin and lysozyme secreted from the cultured cell were measured by immunohistochemical study and dot blotting. The level of mucin genes 5AC (MUC5AC), MUC5B, MUC8 messenger RNA(mRNA)s and the level of lysozyme mRNA were measured on each culture period by reverse transcription (RT)-polymerase chain reaction (PCR). RESULTS: Ciliogenesis usually started on the 16th-18th day after confluence. The percentage of ciliated cells increased over time up to 10.6% but that of secretory cells remained at about 40% throughout culture duration. By the 14th day after confluence, the amounts of mucin and lysozyme secretion increased rapidly and then maintained a plateau. The expression levels of MUC5B, MUC8 and Lysozyme increased with culture time. Especially, MUC8 showed a dramatic increase on the 28th day after confluence. In contrast, the level of MUC5AC mRNA showed a peak on the 14th day after confluence, and then decreased. CONCLUSION: Ciliary differentiation of NHMEE cells can be induced by ALI culture system. Our study also suggests that secretory function develops earlier than ciliogenesis, and that the expressions of MUC5B and MUC8 mRNAs increase as a function of differentiation.

Secretory Differentiation of Serially Passaged Normal Human Middle Ear Epithelial(NHMEE) Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The purpose of this study was to subculture normal human middle ear epithelial (NHMEE) cells, investigate whether the subcultured NHMEE cells could have ability to differentiate into secretory cells, and establish a method to get cultured NHMEE cells for further study of human middle ear epithelial differentiation and secretion. MATERIALS AND METHOD: Freshly isolated epithelial cells from healthy middle ear mucosa were subcultured repeatedly after enzymatic disaggregation in serum-free medium on plastic tissue culture dishes. The subcultured cells were counted after every passage and tested for secretory differentiation in air-liquid interface (ALI) cultures. The apical secretion of cultured NHMEE cells were characterized by immunoblotting and Western blotting. RESULTS: Attachment rate of subcultured NHMEE cells was over 70% through every passage. Cells proliferated by 22 fold from passage-1 to passage-2 (P-2), but passage-4 cells did not proliferate. P-2 NHMEE cells in ALI cultures was stained with mucin antibody (H6C5) but not b-tubulin antibody. Cultured NHMEE cells secreted mucin and lysozyme. CONCLUSION: P-2 NHMEE cell cultures retained many important features of normal epithelium and were suitable for conducting many studies of human middle ear epithelial cell biology including cell differentiation and secretion.

Establishment of Normal Human Middle Ear Epithelial (NHMEE) Cell Using Serum-free Media / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Despite many reports about middle ear mucosal epithelial cell culture of experimental animals like rat, gerbil or chinchilla, normal human middle ear epithelial (NHMEE) cells have not been cultured in vitro yet. We attempted to culture NHMEE cells and confirm them to be epithelial cells. MATERIALS AND METHOD: Healthy middle ear mucosa was harvested during the otologic surgery without contamination. Specimen was cultured by primary explant culture method and proliferating cells were subcultured after enzymatic disaggregation. Cultured cells were observed using phase contrast light microscope, scanning electron microscope and transmission electron microscope. Immunocytochemical stain was performed to identify the expression of cytokeratin, vimentin and von Willebrand factor. RESULTS: Cultured cells were of polygonal shape and the cell surfaces were covered by microvilli. The immunocytochemical stain revealed cytokeratin in all the cultured cell, whereas vimentin was co-expressed in some of the cells and von Willebrand factor was not expressed at all. We could observe desmosome or tonofilament in cultured cells by TEM. Although cells proliferated 20 or 30 fold in every passage, the passage-4 cells did not proliferate and many vacuoles were formed. CONCLUSION: The NHMEE culture system using serum free media will provide a good model for the study of human middle ear epithelial differentiation and secretion.