100 Co-Culture of Human type I and type II Pneumocyte Cell Lines as a Model of Alveolar Epithelium for Toxicity Testing

Abstract The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are thus of research interest in studying respiratory exposure to therapeutic and hazardous materials such as plastic particles which represent a new threat for human health. There is a need therefore to develop sophisticated models of the human alveolar epithelium, which better represent the different cell types present in the native lung. Our aim was to develop an air-liquid interface model of the alveolar epithelium by incorporating human cell-lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. Working both in monotypic cultures and cocultures, we compared the morphology of single cells and the structure of cell layers of the two cell-lines using light and electron microscopy. We measured barrier function by trans-epithelial electrical resistance (TEER). We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. We demonstrated that barrier properties can be maintained for 30 days. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung (Brookes (2021) PLoSONE 16(9):e0248798). In summary, our results support the coculture of these two cell-lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium. Funding: From the European Union’s Horizon 2020 research and innovation programme under grant agreement No965367 (PlasticsFate)