In Vitro Model of Invasive Pulmonary Aspergillosis in the Human Alveolus
Cellular bilayer models can be used to simulate many biological compartments. Here, we describe a cell culture model of the human alveolus that enables the study of early invasive pulmonary aspergillosis. The cellular bilayer is constructed with human alv
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1. Introduction Transwells® are semipermeable synthetic membranes that enable the growth of various cell types and the simulation of biological compartments. Here, we describe a cellular bilayer grown on a polyester insert that mimics the alveolar-capillary barrier. This bilayer model can be used to study many aspects related to the pathophysiology and pharmacology of invasive pulmonary aspergillosis. This bilayer model was originally developed to investigate the pathogenesis of Mycobacterium tuberculosis (1, 2). The model was subsequently adapted to study Aspergillus fumigatus, including the kinetics of clinically relevant biomarkers, the invasion of hyphae, the effect of antifungal agents and the antifungal effect of immunological effectors (3, 4). Human alveolar epithelial cells are grown on the upper surface of a polyester membrane that is perforated with 3-μm pores. Human pulmonary artery endothelial cells are grown on the under surface. The cellular bilayer defines two compartments: (1) an upper
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_24, © Springer Science+Business Media, LLC 2012
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compartment that simulates the alveolar airspace and (2) a lower compartment that simulates the pulmonary capillary. The alveolar compartment is inoculated with A. fumigatus conidia, where they germinate to form hyphae. These tissue invasive forms penetrate the cellular bilayer, thus mimicking invasion in the human lung. Antifungal agents can be applied to the endothelial compartment to simulate the systemic administration of these compounds. Immunological effectors can be added to the alveolar or endothelial compartment. Invasion can be assessed directly (e.g. by confocal microscopy) or via the measurement of fungal-related biomarkers.
2. Materials Prepare solutions at room temperature and store at 4°C, unless otherwise stated. Media containing Foetal bovine serum (FBS) can be used for up to 1 month. 2.1. Construction of the Cellular Bilayer
1. Human pulmonary artery endothelial cells (HPAECs, see Note 1). 2. EGM-2 BulletKit (medium; see Note 1) is prepared following the manufacturer’s instructions by adding the provided supplements required to support the growth of HPAECs (ascorbic acid, heparin, hydrocortisone, human endothelial growth factor, 2% FBS, vascular endothelial growth factor, human fibroblast growth factor-B and R3-insulin-like growth factor-1) to the supplied basal medium EBM-2 (see Note 1). 3. Human alveolar A549 epithelial cells (see Note 2). 4. EBM-10%: 10% FBS (see Note 1) in EBM-2 basal medium (see Note 1). 5. Hank’s buffered salt solution (HBSS) with phenol red without calcium and magnesium (see Note 1). 6. 0.25% Trypsin-EDTA solution. 7. Transwell® permeable supports (inserts), 3-μm polyester membranes, 6.5-mm (24-well) inserts (see Note 3). 8. Vented T75 and T162 flasks.
2.2. Experimental Components
1. Phosphate buffered saline (PBS). 2. Potato de
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