Immune Responses to Candida albicans in Models of In Vitro Reconstituted Human Oral Epithelium
In this protocol, we describe the application of commercially available three-dimensional organotypic tissues of human oral mucosa to study the interaction between Candida albicans and epithelial cells. Infection experiments show high reproducibility and
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1. Introduction Three-dimensional organotypic tissue models such as reconstituted human epithelium (RHE) have recently become a useful tool with which to study oral epithelial C. albicans infections (1–6). These models of mucosal candidiasis closely parallel the in vivo situation and allow studies of the physiological functions of pathogen virulence factors by using Candida mutant strains (7–11). At least two commercially available oral models are now available (SkinEthic Laboratory and MatTek Corporation). Both are grown at the airliquid interface on microporous membranes in chemically defined medium to form an oral mucosa analogue. The advantages of these models, compared to more complex systems, are the focused analysis of defined conditions and the easier integration of the immune
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_22, © Springer Science+Business Media, LLC 2012
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cells of the skin that are relevant to defence. These days, vaginal and epidermal models can also be purchased from these companies in order to study different host niches. RHE models are therefore a very promising tool to study host/ pathogen interactions, and the effects of antimicrobial substances can be easily tested to give insights into new clinical approaches. Several methods can be successfully used to analyse the infected model. Epithelial cell damage caused by C. albicans can be quantified by lactate dehydrogenase (LDH) detection, cytokine response can be analysed by ELISA, and protein and RNA transcription analysis can be done from a single sample to study signal transduction pathways.
2. Materials (see Note 1 ) 2.1. Establishment of the Oral Candidiasis Model
1. Sabouraud dextrose agar: 1% (w/v) peptone, 4% (w/v) glucose (Dextrose), 1.2% (w/v) agar, 1% (v/v) penicillin (10,000 U/ mL)/streptomycin (10 mg/mL). 2. Phosphate-buffered saline (PBS), cell culture grade. 3. 0.9% NaCl solution. 4. YPD (Yeast extract peptone dextrose) liquid media: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose (dextrose).
2.2. Preparation, Pre-incubation, and Infection of Tissue Model
1. 6-Well tissue culture plate, sterile with lid. 2. Tweezers, sterile. 3. SkinEthic reconstituted human oral epithelium (RHO/S/5), small: tissue surface 0.5 cm2, age day 5, oral head and neck squamous cell carcinoma cell line TR146 (SkinEthic, Nice, France). 4. SkinEthic maintenance medium (SkinEthic) without antibiotics and antimycotics. 5. EpiOral tissue model (ORL-200), small: tissue surface 0.6 cm2, normal human oral keratinocytes (NHOK), non-diseased from adult human oral tissue obtained from patients undergoing tooth extractions (MatTek). 6. MatTek maintenance medium (MatTek) without antibiotics and antimycotics. 7. Surgical disposable scalpels, sterile no. 11 and 21.
2.3. LDH Cell Damage Assay
1. Cytotoxicity Detection Kit (LDH) (Roche Diagnostics). 2. L-Lactate dehydrogenase (L-LDH) (Roche Diagnost
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