Lung microvascular endothelial cells are hypothesized to contribute to alveolar repair and regeneration. We aim to develop an open-top alveolus-on-chip system with vascularized lung endothelial cells and pericytes, cultured in networks, and study interactions with alveolar epithelial cells.

Primary endothelial cells (CD31⁺), pericytes (NG2⁺) and alveolar epithelial type-2 cells (AEC2; HTII-280⁺) were isolated from resected peripheral human lung tissue and expanded in vitro. Cells were seeded in a fibrin-collagen hydrogel inside an open-top alveolus-on-chip (2 mm diameter, 500 µm high culture chamber) to from vascular networks. The open-top design enables controlled co-culture of vascular networks with monolayers of AEC2.

The expanded endothelial cells were analyzed by flow cytometry and shown to be CD31⁺ and CD140b ^-, whereas pericytes were NG2⁺ and CD140b⁺. The endothelial cells (CD31⁺ CD144⁺) formed vascular networks by themselves, as well as in co-cultures with pericytes (NG2⁺) and in co-cultures with MRC-5 fibroblasts (?-SMA⁺) at day 5. Endothelial cells alone were able to form networks with typical vessel diameters of 5 to 10 ?m compared to 1 to 3 ?m in networks formed with pericytes. In presence of pericytes, a more continuous endothelial network was observed when compared to co-cultures with fibroblasts, while the vessel diameters were comparable in both conditions. Monolayers of AEC2 (surfactant protein C⁺) were successfully cultured at day 5 on top of the vascular networks until day 12.

This open-top alveolus-on-a-chip model enables controlled studies of the role of vascular networks in alveolar repair, e.g. upon exposure to cigarette smoke.