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Development of a human lung model system for the identification of chemical respiratory allergens
Introduction
No validated methods exist for the evaluation of chemicals inducing respiratory allergy, neither in vivo nor in vitro. Therefore, the development of tests is of great concern, especially considering the new legislation of chemicals by the European Union (REACH). Attention should be directed to in vitro models because the REACH guidelines clearly state that tests on animals should be avoided, and a complete ban on animal testing for cosmetic ingredients will be implemented in Europe by 2013.
To establish an in vitro test system, we developed an immunocompetent, three-dimensional co-culture system representing the proximal alveolar region of the human lung. The model will be exposed to respiratory sensitizers or irritants in the form of aerosols or particles at the air/liquid interface mimicking the in vivo situation in the lung as closely as possible.

METHODS
Three types of co-culture systems were established:
Double cell co-culture model:
• Human lung epithelial cells (ECs): A549 cells
• Human alveolar macrophages (AMs): U937 cells, differentiated to AMs with phorbolmyristateacetate (PMA)
Triple cell co-culture model, type 1 and type 2:
• ECs: A549 cells
• AMs: U937 cells, differentiated to AMs with PMA
• Human immature dendritic cells (DCs): DCs differentiated from human peripheral blood monocytes

ECs were grown on BD Falcon cell culture inserts for 7 days. After formation of the epithelial cell layer, AMs were seeded on top of the ECs. For the triple cell co-culture model type 1, DCs were cultured in the cell culture well (Fig. 1A); for the triple cell co-culture model type 2, DCs were seeded on the basal side of the insert (Fig. 1B).

   Figure 1: A, B: Co-culture models, C: VITROCELL ® VAGF Nebulizer, D: Exposure module

Endpoints for characterization of the cell cultures:
• Integrity of the EC layer: Measurement of the transepithelial electric resistance (TEER) with Millicel-ERS (Millipore).
• Functionality of the EC layer: Staining of surfactant protein (proSP-C)
• Immature DCs: Staining of DC-marker CD1a and maturation markers CD83/CD86

Well-known respiratory sensitizers and respiratory irritants will be tested with all cell co-culture models using the Cultex® in vitro inhalation system (Fig. 1 C, D). Aerosols or particles will be generated with the VITROCELL® VAGF Nebulizer (Fig. 1C).

RESULTS
TEER measurement: Electrical resistance of inserts cultured with ECs increased over time. Since highest TEER values were reached on day 7 (red
arrow), inserts were processed at that time (Fig. 2).

Figure 2: TEER measurement. Each value represents the mean and standard deviation from 3 individual inserts cultured with ECs (3 measurements per insert).

Staining of surface markers: Production of the surfactant protein proSP-C in ECs was shown with immunocytochemistry (Fig. 3A). As a negative control, AMs did not stain positive for proSP-C (Fig. 3B). The immature status of DCs was analyzed with immunocytochemistry (Fig. 3C) and flow cytometry (Fig. 3D). Both methods demonstrated that the DC cultures were highly CD86/CD83-negative. Flow cytometry additionally showed that the cells were positive for the DC-marker CD1a (Fig. 3D).
CD1a FITC
 
Figure 3: Immunocytochemistry (A-C) and flow cytometry (D) of surface markers. A, B: ECs (A) and AMs (B) were stained with DAPI (blue signal) and anti-proSP-C (red signal). ProSP-C was detected on ECs, but not on AMs. C: DCs were stained with DAPI (blue signal) and CD86 (red signal). Most cells were CD86-negativ (arrowheads). A CD86-positive cell can be seen in the middle (arrow). D: DCs were stained with CD83/CD1a or CD86/CD1a and analyzed with flow cytometry. Most cells stained CD83/CD86-negative and CD1a positive (green population).

DISCUSSION
Due to the implementation of a complex three-dimensional co-culture system, we hope to identify biomarkers for respiratory sensitization. Since
the in vitro exposure technique can also be performed with powdered solids, and therefore water solubility of the test substance is not mandatory, the method should be applicable for a wide range of chemicals.

ACKNOWLEDGEMENTS
Thanks to Sabine Maier for performing immunocytochemistry and to Christopher Kaltenecker for TEER measurements and cell culture work.
MACS Technology for cell separation
New inhalation exposure system—Multipurpose Inhalation Cage Unit