Using a Novel Microfabricated Model of the Alveolar-Capillary Barrier to Investigate the Effect of Matrix Structure on Atelectrauma

Abstract

The alveolar-capillary barrier is composed of epithelial and endothelial cells interacting across a fibrous extracelluar matrix (ECM). Although remodeling of the ECM occurs during several lung disorders, it is not known how fiber structure and mechanics influences cell injury during cyclic airway reopening as occurs during mechanical ventilation (atelectrauma). We have developed a novel in vitro platform that mimics the micro/nano-scale architecture of the alveolar microenvironment and have used this system to investigate how ECM microstructural properties influence epithelial cell injury during airway reopening. In addition to epithelial-endothelial interactions, our platform accounts for the fibrous topography of the basal membrane and allows for easy modulation of fiber size/diameter, density and stiffness. Results indicate that fiber stiffness and topography significantly influence epithelial/endothelial barrier function where increased fiber stiffness/density resulted in altered cytoskeletal structure, increased tight junction (TJ) formation and reduced barrier permeability. However, cells on rigid/dense fibers were also more susceptible to injury during airway reopening. These results indicate that changes in the mechanics and architecture of the lung microenvironment can significantly alter cell function and injury and demonstrate the importance of implementing in vitro models that more closely resemble the natural conditions of the lung microenvironment.

Figure 1

(A) Schematic representation of the micro-nano biomimetic system used in this study. (B) Image of PDMS microfluidic chamber, (C) SEM image of SU8-2075 spin coated photoresist layer with an average thickness of 200 µm, and (D) schematic representation of occluded airway reopening simulation for epithelia/endothelial cells co-cultured on opposite sides of the fiber meshes.

Figure 2

Tensile test results for wet nano fiber meshes prepared using different PCL-to-gelatin rations. (A) Representative stress vs. strain curves for the different fiber mesh compositions used in this study and (B) Computed Young Modulus values. *Indicates significant difference between groups (p-value ≤ 0.009).

Figure 3

(A) Fiber size distribution for fibers fabricated with different PCL/Gelatin ratios, and (B) SEM images showing detail of fiber architecture of representative fiber groups (scale bars: 25 µm). *Indicates significant difference with respect to other groups (p-value < 0.001).

Figure 4

(A) Schematic representation of co-culture system and images of assembled micro-nano fluidic device with co-cultured cells, and (B) confocal images of epithelial and endothelial cells co-cultured on a 75/25 (PCL/gelatin) fiber mesh (Red: cell nuclei stained with Propidium Iodide (PI)/Rnase, and Green: actin cytoskeleton stained with Alexa Fluor 488 Phalloidin).

Figure 5

Actin staining (green) and cell nuclei (blue) of (A) epithelial (A549) and (B) endothelial (HUVEC) cells cultured on PCL (right) or 50/50 (PCL/gelatin) (left) fibers. Scale bar 25 µm.

Figure 6

(A) tight junctions staining (ZO-1: red, Occludin: green, nuclei: blue) of epithelial and endothelial cells co-cultured on PCL (right) or 50/50 (PCL/gelatin) (left) fibers (scale bars: 25 µm). (B) Characterization of ZO-1 and occludin relative expression, and (C) transport of 70 KDa FITC-labeled dextran across co-cultured epithelial/endothelial cells after 60 minutes (2-day co-cultures) (mean ± SEM$)$. *Indicates significant difference with respect to No-cells (control) group (p =  < 0.029).

Figure 7

Airway reopening simulations for epithelial/endothelial cells co-cultured on 50/50, 75/25, and 100/0 (PCL/gelatin) fiber meshes after simulation of airway reopening. (A) Live/dead fluorescence imaging of both A549 and primary human small airway epithelial cells after 5 reopening events and (B) quantification of percentage of cell death after propagation of 5 air bubbles for A549 and primary epithelial cells (mean ± SEM) (scale bars: 150 µm). *Indicates significant difference between groups (p-value ≤ 0.018).