A TRIPLE CELL CO-CULTURE MODEL OF THE AIR-BLOOD BARRIER RECONSTRUCTED FROM PRIMARY HUMAN CELLS.

Reliable in vitro human models for investigating airway toxicity are needed to advance Tox21 efforts to replace animal testing with human-based model systems. In vitro airway models based on primary human cells have been described, however, models based on animal cells or human cell lines are still most commonly employed. In the present work, we describe development of an in vitro air-blood barrier model derived from primary human alveolar epithelial cells, pulmonary endothelial cells and monocyte-derived macrophages. The triple co-culture model was characterized by histological and immunohistochemical methods and transmission electron microscopy. Barrier formation was assessed by measurement of transepithelial electrical resistance (TEER). Confocal imaging demonstrated epithelial staining for cytokeratin 19 as well as tight junction proteins ZO-1 and occludin, while the endothelial cell layer stained positive for von Willebrand factor and e-cadherin. CellTracker dye was used to visualize macrophages on the luminal side of the alveolar cultures. The model developed peak TEER of > 1,000 Ω * cm2 within 9-12 days, and maintained TEER > 400 Ω * cm2 for up to 30 days. This new human model system shows promise as a useful tool for in vitro airway toxicity investigations.

Air-blood barrier model Alveolar tissue model alveolar/endothelial co-culture BCRP1 carboxypeptidase M CellTracker dye cytokeratin 19 drug transporters e-cadherin  efflux drug transporters macrophages MDR1 MRP1 MRP2 Occludin OCTN1 OCTN2 surfactant protein C Tight junction proteins von Willebrand factor ZO-1