Clinical and Experimental Otorhinolaryngology Vol. 1, No. 2: 86-91, June 2008 DOI 10.3342/ceo.2008.1.2.86 Original Article Establishment and Characterization of an In Vitro Model for Cholesteatoma Alexander M. Raynov, MD1 Yun-Hoon Choung, MD Hun Yi Park, MD Seong Jun Choi, MD Keehyun Park, MD, PhD 1Department of Otorhinolaryngology, University Hospital Tzaritza Joanna, Medical University - Sofia, Sofia, Bulgaria; Department of Otolaryngology, Ajou University School of Medicine, Suwon, Korea Objectives. Experimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a “skin equivalent” and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells. Methods. Primary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry. Results. Different protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts. Conclusion. 3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue. Key Words. Cholesteatoma, In vitro, Keratinocytes, Culture INTRODUCTION Middle ear cholesteatoma is characterized by keratinocytic hyper- proliferation with bony destruction and granulation tissue by chron- ic inflammation. The pathogenesis of middle ear cholesteatoma has been debated for a century, but there are four basic theories: invagination, basal cell hyperplasia, epithelial ingrowth and squa- mous metaplasia. During last century, etiopathogenicity of middle ear cholestea- toma raised many controversies and still a unanimous concept doesn’t exist. From a histological point of view this pathological tissue is composed of 2 integrated parts - matrix and perimatrix. Received November 13, 2007 Accepted after revision February 10, 2008 Corresponding author : Keehyun Park, MD, PhD Department of Otolaryngology, Ajou University School of Medicine, San 5 Woncheon-dong, Yeongtong-gu, Suwon 443-721, Korea Tel : +82-31-219-5000, Fax : +82-31-219-5264 E-mail : parkkh@ajou.ac.kr This study was presented at the 8th International Conference on Cholesteatoma and Ear Surgery which was held in Antalya, Turkey through June 15-20, 2009, and received the cholesteatma prize. 86 The interaction between cellular components is essential for the major histopathological features of cholesteatoma - hyperprolif- eration and bone destruction. In 1964, the American anatomist Gray referred to middle ear cholesteatoma as “skin in the wrong place” (1). This simplified misdefinition stuck into literature and has been cited profusely in many scientific papers. The metapla- sia theory (2) and existence of so-called “congenital” cholesteatoma put into question the “skin origin” of middle ear cholesteatoma and gave etiopathogenetical evidences against the simplified definition “skin in the wrong place”. The list of histological dif- ferences includes the absence of cutaneous adnexa and special- ized dermal structures. Neither hypodermal structures, nor blood vessels, or nerve endings have been ever described in choleste- atoma. The only similarity that is left between them is the pres- ence of multistratified squamous epithelium, but in cholesteatoma this epithelium is with hyperproliferative properties. Experimental models are critical for studying pathogenesis of middle ear cholesteatoma. In vivo models were tried to establish by chemical injection into the middle ear (3), ligation of exter- nal auditory canal (4) and transplantation of skin (5). Keratinocytes of middle ear cholesteatoma were cultured by tissue culture (6),