Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media

The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies

Multi-scale spatial heterogeneity enhances particle clearance in airway ciliary arrays

Mucus clearance constitutes the primary defence of the respiratory system against viruses, bacteria and environmental insults [1]. This transport across the entire airway emerges from the integrated activity of thousands of multiciliated cells, each containing hundreds of cilia, which together must coordinate their spatial arrangement, alignment and motility [2, 3]. The mechanisms of fluid transport have been studied extensively at the level of an individual cilium [4, 5], collectively moving metachronal waves [6-10], and more generally the hydrodynamics of active matter [11, 12]. However, the connection between local cilia architecture and the topology of the flows they generate remains largely unexplored. Here, we image the mouse airway from the sub-cellular (nm) to the organ scales (mm), characterising quantitatively its ciliary arrangement and the generated flows. Locally we measure heterogeneity in both cilia organisation and flow structure, but across the trachea fluid transport is coherent. To examine this result, a hydrodynamic model was developed for a systematic exploration of different tissue architectures. Surprisingly, we find that disorder enhances particle clearance, whether it originates from fluctuations, heterogeneity in multiciliated cell arrangement or ciliary misalignment. This resembles elements of 'stochastic resonance' [13-15], in the sense that noise can improve the function of the system. Taken together, our results shed light on how the microstructure of an active carpet [16, 17] determines its emergent dynamics. Furthermore, this work is also directly applicable to human airway pathologies [1], which are the third leading cause of deaths worldwide [18]