Air-Liquid interface cultures to model drug delivery through the mucociliary epithelial barrier

Abstract

Epithelial cells from mucociliary portions of the airways can be readily grown and expanded in vitro. When grown on a porous membrane at an air-liquid interface (ALI) the cells form a confluent, electrically resistive barrier separating the apical and basolateral compartments. ALI cultures replicate key morphological, molecular and functional features of the in vivo epithelium, including mucus secretion and mucociliary transport. Apical secretions contain secreted gel-forming mucins, shed cell-associated tethered mucins, and hundreds of additional molecules involved in host defense and homeostasis. The respiratory epithelial cell ALI model is a time-proven workhorse that has been employed in various studies elucidating the structure and function of the mucociliary apparatus and disease pathogenesis. It serves as a critical milestone test for small molecule and genetic therapies targeting airway diseases. To fully exploit the potential of this important tool, numerous technical variables must be thoughtfully considered and carefully executed.

Figure 1.. An illustrated view of the multilayer human airway mucosal surface barrier.

A) An overview of the mucociliary escalator system that is critical for innate airway defense. It consists of two essential layers, an overlying mucus layer and the periciliary layer (PCL) that is positioned between the mucus layer and the apical cell borders. B) A higher magnification view of a secretory cell highlighting stored mucins in secretory granules and their secretion contributing to the mucus layer. C) A 3D rendering of atomic force microscopy (AFM) topography of gel forming mucins, MUC5AC and MUC5B, the major macromolecules in the mucus layer showing their infrastructure to form a protective net-like gel layer. D) A magnified view of the PCL constituted by cilia and microvilli, both of which are densely decorated with membrane bound mucins, MUC1, MUC4, and MUC16. E) An AFM image of the membrane mucins, MUC4 and/or MUC16, isolated from airway epithelial cells, highlighting the presence of keratan sulfate decoration (white arrow). Figure is modified from [20] and [21].

Figure 2.. Insert membrane choice alters ALI culture morphology.

Human tracheobronchial airway epithelial cells grown on polyethylene terephthalate (PET; Snapwell and T-Clear Transwells; Corning) and polytetrafluoroethylene (PTFE; Millicell CM) membranes, demonstrating a thicker and more stratified cell layer on the PTFE membrane. A) Cell layer thickness obtained on both membrane types for samples from nine separate donors. B-C) Photomicrographs of representative histological sections of cells on PTFE (B) and PET (C) membranes. Figure is modified from [138].

Figure 3.. Media choice alters ALI culture morphology and electrophysiology.

A-F) Representative histological sections stained with hematoxylin and eosin (H&E) or Alcian blue-Periodic acid Schiff (AB-PAS) of human tracheobronchial airway epithelial cells (HBECs) in various differentiation media choices as indicated. A-B) UNC ALI; C-D) Vertex ALI; E-F) PneumaCult ALI. Different proportions of characteristic basal, secretory and ciliated cell types, alterations in stored mucosubstances, and variable stratification and epithelial thickness are evident. HBECs were grown on PTFE membranes. All scale bars = 25 μM. G-H) TECC-24 electrophysiology measurements of non-CF HBEC ALI cultures grown in UNC ALI, Vertex ALI, or PneumaCult ALI differentiation media. G) TECC-24 tracing. Acute addition of benzamil (Benz), forskolin (FSK), genistein (Gen), and CFTR inhibitor-172 (CFTRinh-172) indicated by arrows. H) Basal equivalent current (Ieq) and change (Δ) in Ieq in response to Benz, FSK, Gen, and CFTRinh-172. One biological donor; 8 replicates.

Figure 4.. Culture media choice impacts the composition of the tracheobronchial epithelial cell apical secretion proteome.

Among 825 identified apical secretome proteins, 80 and 32 were significantly increased and decreased, respectively in airway epithelial ALI cultures grown in PneumaCult ALI versus UNC ALI media. Each green dot represents a protein with significantly altered signal abundance in the volcano plot.