Clearing Things Up: A Vertical Mucus Clearance Assay to Investigate Rheological and Biochemical Parameters Governing Mucociliary Clearance

Mucociliary clearance (MCC) plays an essential role to protect the airways from pathogens and pollutants. Impaired MCC leads to a host of diseases and initiates a cascade of chronic infection, inflammation, and reduced lung function. Understanding the mechanisms of clearance is a critical step in preventing and ultimately treating disease. The fundamental mechanisms guiding clearance are still poorly understood. Existing models have focused on fluid rheology, but haven't reached a consensus on which parameters govern the system. In this work I build an in vitro model capable of evaluating rheological and biochemical parameters and their effect on clearance.;My model integrates the air-liquid interface (ALI) bronchial epithelial cell culture model with microfluidics to create a mucus clearance assay (MCA). Growing well-ciliated cells inside of a fluidics channel allows me to use external flow to investigate cilia-ASL interactions. I use external flow to build and validate a model of cilia hydrodynamics. Then I demonstrate external flow can be used as a force probe to investigate adhesion on the epithelial surface.;Next I integrate the MCA with a tilting microscope to explore the effects of gravity on mucociliary clearance. Gravity has been largely ignored in mucus clearance experiments despite vertical transport in vivo and therapies exploiting gravitational drainage. I observe fluids transporting horizontally, but failing to transport when the cilia driven flow is opposed by gravity. This establishes gravitational effects as a necessary requirement for evaluating the parameters that govern MCC. Next I tested a 4.5% 1MDa PEG solution that matched the apparent viscosity of mucus and observed that unlike mucus, the PEG solution didn't transport vertically. This experiment demonstrates that viscosity alone is not a predictor of vertical transport. Finally I suggest that the inability to transport vertically is a result of the system dehydrating and posit that mucus' ability to transport vertically is the result of biochemical adhesion with the cilia.