An in vitro model of murine middle ear epithelium.

Disease models & mechanisms (2016-09-24)
Apoorva Mulay, Khondoker M Akram, Debbie Williams, Hannah Armes, Catherine Russell, Derek Hood, Stuart Armstrong, James P Stewart, Steve D M Brown, Lynne Bingle, Colin D Bingle
ABSTRACT

Otitis media (OM), or middle ear inflammation, is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology it is clear that epithelial abnormalities underpin the disease. There is currently a lack of a well-characterised in vitro model of the middle ear (ME) epithelium that replicates the complex cellular composition of the middle ear. Here, we report the development of a novel in vitro model of mouse middle ear epithelial cells (mMECs) at an air-liquid interface (ALI) that recapitulates the characteristics of the native murine ME epithelium. We demonstrate that mMECs undergo differentiation into the varied cell populations seen within the native middle ear. Proteomic analysis confirmed that the cultures secrete a multitude of innate defence proteins from their apical surface. We showed that the mMECs supported the growth of the otopathogen, nontypeable Haemophilus influenzae (NTHi), suggesting that the model can be successfully utilised to study host-pathogen interactions in the middle ear. Overall, our mMEC culture system can help to better understand the cell biology of the middle ear and improve our understanding of the pathophysiology of OM. The model also has the potential to serve as a platform for validation of treatments designed to reverse aspects of epithelial remodelling that underpin OM development.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Retinoic acid, ≥98% (HPLC), powder
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, lyophilized powder, Protein ≥85 %, ≥400 Kunitz units/mg protein
Sigma-Aldrich
Cholera Toxin from Vibrio cholerae, ≥90% (SDS-PAGE), lyophilized powder
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide hydrate, ≥96.5% (HPLC), ≥96.5% (spectrophotometric assay), from yeast
Sigma-Aldrich
apo-Transferrin human, powder, BioReagent, suitable for cell culture, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Insulin from bovine pancreas, γ-irradiated, BioXtra, suitable for cell culture, potency: ≥20 units/mg (USP units), lyophilized powder
Sigma-Aldrich
p-Xylene-bis(N-pyridinium bromide), ≥95% (TLC)