Exploring Ciliary Dynamics: Assessing Motility and Induced Velocity in Human Airway Cell Cultures

Abstract

The human respiratory tract going from the nose to the terminal bronchioles is lined by a mucociliated epithelium. Here, cilia underpin an essential defence mechanism known as MCC. This process clears inhaled pathogens and contaminants from the airways and exhibits various degrees of complexity. Differently from the case of microorganisms, where cilia are often distant more than a few micrometers, in the airway epithelium they are significantly packed within a cell. Due to the high cilia density, steric interactions between cilia are thus possible. Moreover, cilia are immersed in a non-Newtonian fluid, whose elastic modulus plays an important role for the synchronisation and are tethered by mucins that provide further mechanical coupling between them. Thirdly, cilia are also structurally connected through the actin mesh that links elastically all cilia of the same cell at the base. The current model of MCC suggests that particles are trapped in the top viscous mucous layer of the ASL and propelled outside the airways through a unidirectional fluid flow driven by cilia, beating in the underlying PCL. The ASL not only serves as a protective barrier, but also contributes to maintain normal respiratory mechanics, providing a favourable fluid environment for ciliary beating and allowing ciliary alignment and synchronisation via hydrodynamic coupling. Even though ciliary molecular structure is well understood, the question of how motile cilia interact with one another and with the complex surrounding fluid is still an open and pressing one, with immediate implications for the medical community. In vitro models of human airway epithelium are an effective tool to study MCC and disease pathogenesis in the respiratory tract. However, depending on the cell origin, culture medium and growth factors employed, these tissues may differ for the amount and type of cells. Therefore, number, distribution and ciliary length, and physical properties of the ASL are often highly diverse among the samples, leading to a variability in CBF, coordination and alignment. Hence, the need to measure coverage in motile cilia and CBF in in vitro mucociliated tissues in order to quantify physical differences between different cells and different culture protocols and be able to correlate them to the MCC efficiency. Video microscopy can be used to characterise these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the CBF of very small areas, while requiring human intervention and training for their use. In the human airway epithelium, mucus transport can be measured experimentally but the complex structure of the PCL, occupied by cilia-tethered mucins which create a brush with nanometric mesh size, is more challenging as for example it interferes with the conventional use of tracer beads. Earlier studies have measured the average displacement of fluorescent dyes localised in the PCL but, having photoactivated the compound in columns perpendicular to the epithelium, did not manage to extract a clear velocity profile in this layer. Furthermore, substantial research has been devoted to understanding cilia-driven mucus transport from a theoretical standpoint. The physical and numerical complexity of modeling fluid flow at the scale of individual cilia, however, has proven to be an arduous challenge. Given these obstacles, new experimental methods are required to overcome the constraints posed by the PCL structure in determining how the liquid trapped in this brush is propelled by the cilia. This thesis details significant advancements in the use of video microscopy for the analysis of high speed videos of in vitro human airway mucociliated tissues, specifically targeting (1) epithelial properties such as cilia distribution and beating frequency on a tissue scale, and (2) fluid dynamics in the PCL at the cilium scale. Chapter 1 provides an overview of the biological and mechanical properties of motile cilia, emphasizing their role in the respiratory system and the critical questions that drive this research. Chapter 2 details the methods used in our studies. Chapters 3 and 4 introduce a novel, open-source method that automates the full characterization of cilia beating frequency and motile cilia coverage, requiring no manual intervention. The developed algorithm is able to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterise the CBF of all moving areas. This capability is critical for accurately assessing ciliary function across different areas and conditions. Chapter 5 outlines a novel experimental approach developed to navigate the complexities of the PCL structure. This method employs high-speed videos of bronchial cilia beating in a Newtonian fluid, complemented with measurements of the fluid transport in the PCL probed by means of a caged dye. The results from this experimental approach are compared with simulations of ciliary arrays, providing a robust framework for understanding the mechanics of how cilia-driven fluid is propelled within this densely structured environment.