Tracking ciliary development in three-dimensional airway organoids with reversed bipolarity with epi-mode 3D quantitative phase imaging

Cilia are hair-like cellular projections that facilitate movement along many cellular surfaces, including lung epithelia. Ciliary disruption along lung epithelia impairs mucociliary clearance, preventing adequate expulsion of mucus from the airways. In respiratory diseases with known ciliary dysfunction, such as primary ciliary dyskinesia, this mucus accumulation can lead to severe infections, including chronic bronchitis and pneumonia, along the entire respiratory tract. However, their role in other respiratory diseases with similar presentations, such as cystic fibrosis, is not known, as their function cannot be studied in-vivo noninvasively. Airway organoids are three-dimensional cellular cultures that mimic the development and cellular environment of the human lung. However, as most lung organoids mimic the cellular polarity present in the lung, the cilia are encased within the organoid and cannot be easily studied. With the advent of airway organoids with reversed bipolarity (AORB), the cellular organization is flipped, such that the cilia are present on the outer surface of the organoid. Here, we propose using quantitative oblique back-illumination microscopy (qOBM) to study and track ciliary function through AORB development. qOBM is a label-free imaging technique that provides cellular and subcellular resolution of thick-scattering samples. Using qOBM, we characterize ciliary function by regularly imaging healthy AORBs throughout their development. Through this progression, we quantify changes in the density of ciliated cells and their associated ciliary beat frequencies. We aim to compare ciliary function in developing healthy AORBs to ciliary function in developing diseased AORBs, such as cystic fibrosis and chronic lung allograft dysfunction.