Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections.

Chandorkar P, Posch W, Zaderer V, Blatzer M, Steger M, Ammann CG, Binder U, Hermann M, Hörtnagl P, Lass-Flörl C, Wilflingseder D.
Source: Scientific Reports
Publication Date: (2017)
Issue: 7(1): 11644
Research Area:
Dermatology/Tissue Engineering
Respiratory Research
Cells used in publication:
Epithelial, bronchial (NHBE), human
Species: human
Tissue Origin: lung
Epithelial, Small Airway, human (SAEC)
Species: human
Tissue Origin: lung

NHBE or SAE cell culture. Primary normal human bronchial epithelial (NHBE) or small airway (SAE) cells were obtained from Lonza. 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). 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. Cultures were maintained in a humidifed atmosphere with 5% CO2 at 37 °C and then transferred to ALI culture. Te epithelium was fed with B-ALI or S-ALI diferentiation medium (Lonza). The number of days in development was designated relative to initiation of ALI culture, corresponding to day 0. Cells were used between day 50 to day 70 in ALI and under perfusion.

TEER values were measured using EVOM voltohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). Measurements on cells in ALI culture were taken immediately before the medium was exchanged. For measurements, 0.5ml and 1.0ml of medium were added to the apical and basolateral chambers, respectively. Cells were allowed to equilibrate before TEER was measured. TEER values reported were corrected for the resistance and surface area of the Transwell flters.


To study interactions of airborne pathogens, e.g. 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. 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. 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. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.