A CO-CULTURE MODEL OF THE AIR-BLOOD (ALVEOLAR) 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 model was constructed by seeding endothelial cells on the underside of a microporous membrane. Alveolar epithelial cells were then cultured on the top surface of the membrane. The epithelial /endothelial co-culture was continued until barrier development occurred. Finally, monocyte derived macrophages were seeded onto the apical surface of the model. The triple cell 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). Expression of drug transporter genes including ABC family efflux transporters BCRP, MRP1 and MRP2, and organic cation uptake transporters OCTN1, OCTN2 and OCT3 was demonstrated by RT-PCR. Macrophage purity was > 95% (CD14+) as assessed by flow cytometry. Confocal imaging demonstrated epithelial staining for cytokeratin 19 and carboxypeptidase M (alveolar type 1 cell markers), 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 x cm2 within 9-12 days, and maintained TEER > 400 Ω x cm2 for up to 30 days. This new human model system shows promise as a useful tool for in vitro airway toxicity and drug-drug interaction investigations.

ABC efflux transporters, Air-blood barrier, Alveolar tissue (ALV-100), alveolar type I (ATI) epithelial cells, BCRP, carboxypeptidase M, CellTracker dye, cytokeratin 19, e-cadherin, Epithelial /endothelial co-culture, Lung model, MRP1, MRP2, Occluding, OCT3, OCTN1, OCTN2, organic cation uptake, transporters, RAGE, surfactant protein C, TEER, tight junction proteins, von Willebrand factor, ZO-1