88 Clinical and Experimental Otorhinolaryngology Vol. 1, No. 2: 86-91, June 2008 Primary mouse monoclonal anticytokeratin antibody (Sigma) at concentration 1:20 was incubated overnight at RT. Vectorlab Elite ABC kit (Vector Laboratories, Burlingame, CA, USA ) was used for detection of positive reaction using diaminobenzidine substrate. RESULTS Cell culture Enzymatic disaggregation protocol yield a high number of sepa- rated cells. The low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure. As an inflammatory tissue, cholesteatoma posed some difficulties in isolation of primary keratinocytes, because of the attendant inflammation and possible contamination, which requires an adequate decontamination. The first primary keratinocytes appeared usually 7-10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence. The individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient. For instance, attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger prolif- erative ability. Another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro. The above-mentioned dependencies were not statistically significant. Primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern (Fig. 2A). In passage 2 an abrupt deceleration in proliferation was established, accom- panied by morphological alterations such as enlargement and flattening of the cells, as well as vacuolization of the cytoplasm. In passage 3 cells were practically incapable of proliferation. Pri- mary cholesteatoma fibroblasts were steadily maintained with- out significant changes of doubling time until passage 10. Primary cholesteatoma fibroblasts displayed typical spindle-shaped mor- phology upon confluence (Fig. 2B). No spontaneous transforma- tion was detected in any of the cultures. Fig. 2. (A) Confluent monolayer of primary middle ear cholesteatoma (passage I) keratinocytes shows a polygonal appearance. (B) Primary middle ear cholesteatoma fibroblasts show typical spindle-shaped morphology (passage 1) upon reaching a confluence. Original magnification ×400. Fig. 3. Light microscopic observation on 3D-in vitro middle ear cholesteatoma model developed without fibroblasts (A) and in the co-cultured sys- tem with middle ear cholesteatoma fibroblasts (B). Middle ear cholesteatoma keratinocytes show bigger stratification and increased cellular size in the presence of fibroblasts. Hematoxylin-eosin staining, Original magnification ×400. A A B B