Assessment of the feasibility to develop a fast and easy reproducible 3D bronchial model growing at the air-liquid interface: Which critical culture parameters must be controlled?

The discovery and evaluation of new active drugs will be easier thanks to the development of 3D-epithelial model mimicking biological barriers. These models close to in vivo conditions should help to speed up the drug development process while reducing in vivo animal testing. Regarding lung administration, despite many studies exploring the development of 2D-model using Calu-3 cells, there are still multiple disparities about experimental methods and a standardization is needed. We investigated the impact of different culture parameters (time, cell densities, Transwell™ membrane pore sizes or the culture media) on the integrity and the morphology of a 2D model. Using permeability studies, electron microscopy and immunohistochemistry, we propose an easy and reproducible 2D-model with Calu-3 cells fully differentiated after 14 days of growing at the air-liquid interface. Moreover, based on these results, we went further with a 3D-bronchial model which consists of a collagen matrix, fibroblasts laid under the epithelial layer. We aim to assess the feasibility to develop a 3D cell-based model of the human airway growing at the air-liquid interface. Many parameters which have to be tightly controlled to develop an extracellular matrix equivalent without any shrinking of the gel encompassing alive fibroblasts as well as over epithelial cells have been identified. Overall, this in vitro model is a differentiated pseudo-stratified bronchial mucosa with mucus expression which is the basis for further optimization allowing the screening of pulmonary drugs in terms of permeability, cytotoxicity or particle-mucus interactions.