A gel-coated air-liquid-interface culture system with tunable substrate stiffness matching healthy and diseased lung tissues

Abstract

Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into a pseudostratified epithelium. Further, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases.

Bottlebrush Polyethylene Glycol Nanocarriers Translocate across Human Airway Epithelium via Molecular Architecture-Enhanced Endocytosis

“…In the ALI system, HBECs are cultured on a porous plastic membrane, through which nutrients are transported from the cell culture medium on the basal side, whereas on the apical side, cells are in contact with air . After approximately 4 weeks, primary HBECs, or human airway epithelial basal cells, differentiate into ciliated cells and goblet cells, forming a pseudostratified columnar epithelium that recapitulates essential biological features of the human airway epithelium. − Specifically, the pseudostratified airway epithelium consists of three layers: (i) an intact endogenous mucus hydrogel layer, (ii) a periciliary layer that separates the mucus hydrogel from the epithelial cells, and (iii) a layer of epithelial cells connected by cell junctions (Figure A) …”

“… 46 − 49 Specifically, the pseudostratified airway epithelium consists of three layers: (i) an intact endogenous mucus hydrogel layer, (ii) a periciliary layer that separates the mucus hydrogel from the epithelial cells, and (iii) a layer of epithelial cells connected by cell junctions ( Figure 1 A). 50 …”