Background

The healthy airway mucosal barrier represents a powerful innate immune system designed to protect the pulmonary surfaces from the persistent onslaught of inhaled infectious and noxious substances. During times of airway infection, high-velocity airflow (i.e., cough) represents an effective backup mechanism to clear accumulated mucus. However, abnormalities in mucus properties, which characterize several airway diseases, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and asthma, lead to a reduction in airflow-mediated cough clearance (Button et al., 2016).

To fully elucidate why such mucus fails to clear with cough-induced shear forces, it is necessary to understand the biophysical interactions between airway mucus and airway surfaces. The high-speed airflow associated with cough can remove mucus from airway surfaces through at least two distinct mechanisms: 1) “disadhesion”, whereupon the mucus layer is physically detached from the underlying airway surface as a result of overcoming the “adhesive” interactions between mucus and the airway cell surface; and 2) “tearing”, where chunks of mucus are broken off adherent mucus masses as a result of overcoming mucus-mucus cohesive bonds and interactions.

There are various material science approaches, such as “peel” testers, to quantify the strength of the interaction between one material and another. To initiate a test with these devices, the user “connects” one end of the test material to a movable arm, which then measures the force required to peel the material off a stationary surface. However, the challenge with measuring the mucus adhesive and cohesive strength is that mucus is a fragile material with elastic modulus typically < 10 Pa (Hill et al., 2014) therefore making it impossible to directly connect to a peel device. Furthermore, to measure adhesion strength of mucus to the airway epithelial surface, it is necessary to perform studies in a suitable environment to prevent evaporation of the thin (< 100 μm) mucus layer (Matsui et al., 1998). To overcome these limitations, we have developed a novel peel force technique to quantify both the adhesive and cohesive strengths of airway mucus. Key to this technique, described in detail in this protocol, is the use of a mucus-binding mesh which allows for the peeling of the mucus layer off of the airway surface (i.e., adhesive strength) or another a fixed mucus-binding mesh (i.e., cohesive strength) (Figure 1A). The mucus binding mesh utilized in this protocol is made from a standard laboratory cellulose wipe (Kimwipe). These mucus-binding meshes are very strong, flexible, and as anyone who has blown their nose into tissue paper knows, binds very strongly to mucus. Finally, extensive testing has shown that these meshes are not cytotoxic and do not damage the airway epithelial cells that they are laid upon (Button et al., 2018). Below, we show the construction and application of the mucus-binding mesh-based approach to measure mucus adhesion and cohesion, when connected to a peel test device (Figure 1B).

Using the protocol, we have previously shown that both adhesive and cohesive properties of mucus are sensitive to changes in mucus concentration associated with CF and COPD (Button et al., 2018). In addition to understanding the role of diseased mucus on these biophysical properties, the approach described here can also be useful for identifying/screening therapies designed to improve mucus clearance in subjects with muco-obstructive diseases. Additionally, these measurements can be performed on human-derived mucus samples (i.e., expectorated sputum and isolated airway mucus), potentially valuable for assessment of therapeutic intervention and disease progression. Indeed, we have utilized these techniques to assess the effect of several agents (i.e., mucolytics) which can reduce the adhesive and cohesive strength of CF/COPD mucus (Button et al., 2018). Finally, while this protocol is written to describe the measurement of airway mucus properties, this approach should easily be adapted to measuring adhesive/cohesive properties of other mucus samples (GI, cervical, etc.) as well as other fragile biological or synthetic materials.


Figure 1. Overview of the adhesion and cohesion technique. A. Assembly diagram of the systems for measuring mucus cohesion (left) and adhesion (right). B. Picture of the actual peel test device described in this protocol.