Characterization of Primary Cultures of Cholesteatoma-Associated Fibroblasts

Objectives: The aim of this study was to characterize primary cultured fibroblasts derived from cholesteatoma (CHO) tissue to get evidence of their possible role for determining the different biological behavior of this aural pathology. Materials and Methods: Primary cultures of human fibroblasts were obtained from middle ear CHO specimens and from controls of normal human skin collected during surgical procedures. Cells were incubated with anti-vimentin monoclonal antibody, anti-cytokeratins monoclonal antibody and anti-alpha-smooth muscle actin monoclonal antibody. For reactive oxygen species detection, fibroblasts were incubated with 2',7'-dichlorofluorescein diacetate. Immunofluorescence, flow cytometry, confocal, and transmission electron microscopy were carried out. Results: Cholesteatoma-associated fibroblasts (CHO-AFs) were characterized by a higher degree of cytoplasmic complexity, by the expression of alpha-smooth muscle actin and by a greater basal production of reactive oxygen species in comparison with controls, reflecting a more differentiated phenotype consistent with myofibroblasts. Conclusion: It is possible to suggest that the differentiated phenotype of CHO-AFs might be coupled with a more or less aggressive clinical behavior, and hence, these cultures might represent powerful tools for investigating biology and clinical evolution of this disease.