무혈장 배양액을 이용한 사람의 정상 이중점막 상피세포의 배양

Abstract

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.