passaged subculture (7) and air-liquid interface system (8). How- ever, stable experimental models of middle ear cholesteatoma are not established until now. Our study has aimed to create an “adequate” in vitro model, based on the cell-to-cell interaction between keratinocytes and fibroblasts and to assess the grade of homogeneity between cholesteatoma and skin keratinocytes by focusing on the cytokeratin profile. MATERIALS AND METHODS Cell culture Enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed. The biopsy samples were harvested from patients undergoing surgical treatment in the Department of Oto- laryngology, Ajou University Hospital, Korea. An informed con- sent form approved by the Institutional Review Board of Ajou University Hospital was signed by each patient. Cholesteatoma keratinocytes were cultured in Defined Keratinocytes Serum Free Medium (GIBCO, BRL, Gaithersburg, MD, USA) supplied with growth supplement and 5 mg/mL Gentamicin (GIBCO, BRL) according to the manufacture instructions. Cholesteatoma pri- mary fibroblasts were successfully cultured in Dulbeco Modified Eagle Medium (GIBCO, BRL) supplemented with 10% Fetal Bovine Serum (FBS, Clonetics, Walkersville, MD, USA) and 1% Penicillin-Streptomycin mixture (GIBCO, BRL). Primary ker- atinocytes (passage 1) and fibroblasts (between passages 1-5) were used for establishment of the co-cultured system. HaCaT cell line was kindly provided from the Department of Dermatology in Ajou University Hospital. Feeder layer from cholesteatoma fibroblasts was developed using pretreatment with Mitomycin C (Sigma, St. Louis, MO, USA) in concentration 0.25 g/mL. Fibro- blasts were incubated overnight at 37℃, 5% CO2 in humidified atmosphere. On the next day the media was exchanged and the cells were incubated additionally for another 24 hr. The feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in Transwell (Costar, Cam- bridge, MA, USA) system to develop a subconfluent feeder layer. Feeder layer was incubated for 24-48 hr prior co-cultur- ing. Cholesteatoma keratinocytes and HaCaT cells were seeded MECh keratinocytes MECh fibroblasts A Raynov AM et al.: In Vitro Model for Cholesteatoma 87 at density 2×105 cells/mL over the feeder layer, co-cultured for 3-5 days and air-exposed for a period of 14 days. Two alterna- tive models were elaborated using primary cholesteatoma ker- atinocytes and HaCaT cells (Fig. 1). The control group was pre- pared without feeder layer. Morphological study Transwell membranes were fixed in 4% formaldehyde overnight at 4℃, dehydrated in graded alcohols, cleared in HistoClear (National diagnostics, Atlanta, Georgia) and embedded in paraf- fin. Six m-thick sections were prepared and subjected to rou- tine hematoxylin-eosin staining. Protein profile Polyacrylamide gel electrophoresis has been used for protein separation and staining with Coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and HaCaT cells. Cytokeratin profile After separation, the proteins were transferred to a nitrocellulose membrane. Blocking of nonspecific staining was accomplished with membrane incubation with 5% non-fat skim milk for 1 hr at room temperature (RT). Membranes were then incubated with primary mouse monoclonal anti-cytokeratin antibody clone K 8.12 (Sigma) which is immunospecific for cytokeratin 13 (CK 13) and cytok- eratin 16 (CK 16), with dilution factor 1:100, for 1 hr at RT. After washing membranes were incubated with goat anti-mouse horse- radish peroxidase conjugate (Zymed, San Francisco, CA, USA). The positive reaction was visualized with ECL Western Blotting Detection System (Amersham, Bucks, UK). Immunohistochemistry (IHC) The localization of cytokeratin 13 and 16 in middle ear cholestea- toma tissue was accomplished by IHC study as described before (9). Five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery. Six m thick formalin-fixed paraffin-embedded tissue sections were processed with ABC-method. Samples were deparaf- finized in xylene and rehydrated in alcohol. Heat-induced anti- gen retrieval procedure was applied for antigen unmasking. HaCaT MECh fibroblasts B Fig. 1. Schematic illustrations of air-liquid interface system. (A) Middle ear cholesteatoma (MECh) keratinocytes are co-cultured with fibroblast feeder layer. (B) Alternative model with HaCaT cells is used as a control group. Upper cellular surface is exposed to the air, and the media are supported through the lower semipermeable membrane. This system induces polarity and differentiation.