In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acidsupplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor β (FGF-β) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-β concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Lung cancer is the most frequently diagnosed cancer worldwide and the one that causes the highest mortality. In order to understand the disease and to develop new treatments, in vitro human lung cancer model systems which imitate the physiological conditions is of high significance. In this study, a human 3D lung cancer model was established that features the organization of a tumor with focus on tumor angiogenesis. Vascular networks were formed by co-culture of human umbilical vein endothelial cells and adipose tissue-derived mesenchymal stem cells (ASC) for 14 days in fibrin. A part of the pre-vascularized fibrin gel was replaced by fibrin gel containing lung cancer cells (A549) to form tri-cultures. This 3D cancer model system was cultured under different culture conditions and its behaviour after treatment with different concentrations of tumor-specific therapeutics was evaluated. The evaluation was performed by measurement of metabolic activity, viability, quantification of two-photon laser scanning microscopy and measurement of the proangiogenic factor vascular endothelial growth factor in the supernatant. Hypoxic conditions promoted vascularization compared to normoxic cultured controls in co- and tri-cultures as shown by significantly increased vascular structures, longer structures with a higher area and volume, and secretion of vascular endothelial growth factor. Cancer cells also promoted vascularization. Treatment with 50 µM gefitinib or 50 nM paclitaxel decreased the vascularization significantly. VEGF secretion was only reduced after treatment with gefitinib, while in contrast secretion remained constant during medication with paclitaxel. The findings suggest that the herein described 3D lung cancer model provides a novel platform to investigate the angiogenic potential of cancer cells and its responses to therapeutics. Thus, it can serve as a promising approach for the development and patient-specific pre-selection of anticancer treatment.

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff’s reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.