Generation of a 3D tissue model for the human airway mucosa to study virulence mechanisms of Bordetella pertussis Maria Steinke1,2 | Roy Gross3 | Susanne Bauer3 | Thorsten Walles4 | Heike Walles1,2 1Tissue Engineering and Regenerative Medicine | University Hospital Würzburg | Röntgenring 11 | 97070 Würzburg | Germany 2Fraunhofer Project Group Regenerative Technologies in Oncology | Röntgenring 11 | 97070 Würzburg | Germany | 3Department of Microbiology | University of Würzburg | Am Hubland | 97074 Würzburg | Germany 4Department of Cardiothoracic Surgery| University Hospital Würzburg | Oberdürrbacher Straße 6 | 97080 Würzburg | Germany Introduction Humans are the only natural hosts of Bordetella pertussis, which attacks the airway mucosa and causes whooping cough. In industrial countries whooping cough is resurgent despite broad vaccination coverage, probably due to waning immunity and vaccine-driven evolution. To understand the complex interactions of B. pertussis and its host a species-specific test system for the normal airway mucosa is required. Our aim was to generate such a test system with high in vitro - in vivo - correlation, which is suitable for infection studies with B. pertussis and other human obligate airway pathogens. Methods Generation of the airway mucosa test system To generate a tissue model consisting of a respiratory epithelium and adjacent connective tissue, human tracheobronchial epithelial cells (hTEC) and fibroblasts obtained from surgical specimen were grown on a biological collagen scaffold (BioVaSc) derived from a porcine jejunum segment and cultivated in an airlift environment for three weeks. Infection with B. pertussis Human bronchial segments were incubated with 5 x 108 B. pertussis for 6 h to establish optimal conditions for upcoming infection studies. Untreated biopsies served as controls. Non-destructive Raman micro spectroscopy To rule out tumour-specific cell de-differentiation in tissue biopsies required for tissue model generation, we collected Raman spectra of hTEC and the human airway epithelium adenocarcinoma cell line Calu-3 for comparative analysis. Results Our airway mucosa test system consists of… …a polarized epithelium (A) and vimentin-positive fibroblasts, which migrate into the collagen matrix (B). A H&E After infection of human bronchial segments with B. pertussis we observe… 1 4 3 2 …intracellular bacteria (1), vacuoles and damaged mitochondria (2), destroyed epithelial cells (3) and cell extrusions (4). B Vimentin Raman micro spectroscopy reveals… b 1445-1450 cm-1 1646-1662 cm-1 1073-1087cm-1 relative intensity (a.u.) …mucus-producing goblet cells (C), undifferentiated Cytokeratin 5- (D) and differentiated Cytokeratin 18-positive (E) epithelial cells. a C Alcian blue wave number (cm-1) D Cytokeratin 5 E Cytokeratin 18 100 µm …cell-cell contacts (red box in F), kinocilia (blue box in F), a basement membrane (G, yellow arrow indicates hemidesmosomes) and adjacent collagen fibrils (G, green arrow). F G …good separation of Calu-3 and hTEC datasets after principal component analysis (a). …relevant differences at 1073-1087, 1445-1450 and 16461662 cm-1 (b), which have been described as significant wave number ranges to discriminate human tumour tissue from normal airway and other tissue. Summary We succeeded in generating a 3D test system for the human airway mucosa with high in vitro - in vivo - correlation, which appears suitable for infection studies with B. pertussis and other human obligate airway pathogens. Contact References Steinke M et al. An engineered 3D human airway mucosa model based on an SIS scaffold (2014) 10.1016/j.biomaterials.2014.05.031 Dr. Maria Steinke Tissue Engineering and Regenerative Medicine | Project Group Regenerative Technologies in Oncology University Hospital Würzburg | Röntgenring 11 | 97070 Würzburg | Germany Phone +49 931 31-80720 | fax +49 931 31-80168 [email protected] | [email protected]