Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. To study interactions of airborne pathogens, e.g. TEER values reported were corrected for the resistance and surface area of the Transwell flters. Cells were allowed to equilibrate before TEER was measured. For measurements, 0.5ml and 1.0ml of medium were added to the apical and basolateral chambers, respectively. Measurements on cells in ALI culture were taken immediately before the medium was exchanged. TEER values were measured using EVOM voltohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). Cells were used between day 50 to day 70 in ALI and under perfusion. The number of days in development was designated relative to initiation of ALI culture, corresponding to day 0. Te epithelium was fed with B-ALI or S-ALI diferentiation medium (Lonza). Cultures were maintained in a humidifed atmosphere with 5% CO2 at 37 ☌ and then transferred to ALI culture. Te cells were grown to near confuence in submerged culture for 2–3 days in specifc epithelial cell growth medium according to the manufacturer´s instructions. Te cells were cultured in a T75 fask for 2–4 days until they reached 80% confuence.The cells were trypsinised and seeded onto collagen (Corning collagen I, Rat tail)-coated 0.33 cm2 porous (0.4µm) polyester membrane inserts with a seeding density of 1×105 cells per Transwell (Costar, Corning). Primary normal human bronchial epithelial (NHBE) or small airway (SAE) cells were obtained from Lonza.
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