Clin Exp Otorhinolaryngol.  2008 Jun;1(2):86-91. 10.3342/ceo.2008.1.2.86.

Establishment and Characterization of an In Vitro Model for Cholesteatoma

• Raynov AM
• Choung YH
• Park HY
• Choi SJ
• Park K

• KMID: 1486059
• DOI: http://doi.org/10.3342/ceo.2008.1.2.86

Abstract

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.

Keyword

Cholesteatoma; In vitro; Keratinocytes; Culture

MeSH Terms

Cell Line
Cholesteatoma
Cholesteatoma, Middle Ear
Feeder Cells
Fibroblasts
Immunohistochemistry
Keratinocytes
Keratins
Models, Theoretical
Keratins

Figure

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  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.

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  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.

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  Fig. 3 Light microscopic observation on 3D-in vitro middle ear cholesteatoma model developed without fibroblasts (A) and in the co-cultured system 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.

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  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 fibroblasts. Hematoxylin-eosin staining, Original magnification ×400.

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  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 keratinocytes show different protein profile (arrows) in comparison with HaCaT cells. Blotting with anti-cytokeratin antibody shows the expression of cytokeratin 13 and 16 in both middle ear cholesteatoma keratinocytes and HaCaT cells.

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  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.

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  Fig. 7 Summarized schematic model of putative autocrine and paracrine regulatory mechanisms, responsible for maintaining of middle ear cholesteatoma tissue homeostasis. IL: interleukin; PTHrP: parathyroid hormone-related protein; TGF: transforming growth factor; KGF: keratinocytes growth factor; EGF: epidermal growth factor; GM-CSF: granulocyte-macrophage colony stimulating factor.

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