Raynov AM et al.: In Vitro Model for Cholesteatoma 89 A B Fig. 4. Light microscopic observation on the alternative 3D-in vitro model utilizing HaCaT cells cultured without fibroblasts (A) and air-exposed co-cultured system in the presence of middle ear cholesteatoma fibroblasts (B). HaCaT cells show bigger stratification when co-cultured with fibrob- lasts. Hematoxylin-eosin staining, Original magnification ×400. kDa 250 150 100 75 50 37 23 sm MECh HaCaT A 51 kDa 48 kDa Fig. 6. Immunohistochemical observation on cytokeratin 13 and 16 expression in middle ear cholesteatoma tissue. Cytokeratin 13 and 16 are markers of active proliferative keratinocytes. Immunolabeling shows the expression of them in the whole layer of middle ear cholesteatoma. Air-liquid interface (ALI) Bigger stratification and proliferation have been observed in the proposed 3D in vitro system when primary cholesteatoma ker- MECh HaCaT B Fig. 5. Comparative study on total protein profile (A) and cytokeratin profile (B) between primary middle ear cholesteatoma (MECh) keratinocytes and HaCaT cells. Middle ear cholesteatoma ker- atinocytes show different protein profile (arrows) in comparison with HaCaT cells. Blotting with anti- cytokeratin antibody shows the expression of cyto- keratin 13 and 16 in both middle ear cholestea- toma keratinocytes and HaCaT cells. atinocytes were co-cultured in the presence of fibroblasts (Fig. 3B) in comparison with the control group without fibroblasts (Fig. 3A). Similar morphological landmarks were established in the control group of HaCaT cells (Fig. 4A). Disrupted differentiation in the suprabasal layers was revealed when cholesteatoma ker- atinocytes were air-exposed in the presence of fibroblasts mim- icking the parakeratosis state, which is typical for this patholog- ical tissue in vivo (Fig. 4B). Protein and cytokeratin profile Coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and HaCaT cells revealed signifi- cantly different protein profiles (Fig. 5A). Stronger expression of CK 16 (48 kDa) and CK 13 (51 kDa) was established in cholestea- toma keratinocytes in comparison with HaCaT cells (Fig. 5B). Immunohistochemistry Using mouse monoclonal antibody clone K 8.12, tissue localiza- tion of CK 13 and CK 16 was verified both in the basal and suprabasal layers of cholesteatoma (Fig. 6). The described stain- ing patterns were established in all 5 cholesteatoma samples.