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14 February 2023 Inhalation composition within the mucus layer may have a dual role of either facilitating dissolution of inhaled drug such as corticosteroids but more often than not act- ing as a permeability barrier [51, 52]. e critical fate of inhaled drugs once deposited in the various respiratory regions is therefore often overlooked, resulting in failures of many developmental products in clinical trials reaching the market. Studies such as effects of formulation on epithelial permeability, tight junctions and transport of products through the epithelial layer can be determined using these ALI grown epithelial layers [40, 53]. ese studies can elucidate how the product behaves once depos- ited on the epithelia, by quantitating the levels of the product remaining on top of the epithelia, deter- mining how much product is systematically cleared through the epithelia via paracellular or transcellu- lar mechanisms (Figure 1) and how much permeates inside the epithelial cells. Scaffold-free models e scaffold-free system produces spheroids and organoids, which involves cellular aggregation via the process of organogenesis. e spheroids can be gen- erated by using respiratory cellular models (Figure 1) or extracting cells from lung or transbronchial tissue and then applying diverse techniques, such as non- adhesive surfaces, gravity, centrifugation, constant stirring, electric fields, magnetic force or ultrasound, to produce lung spheroid cells (LSCs). Organoids, on the other hand, are derived from human stem cells and patient-derived, induced, pluripotent stem cells (PSCs) that mimic some specific organ functions. A recent example of this are the lung bud organ- oids that were generated from human PSCs, which developed into branching airway and early alveolar structures after in vivo transplantation [54, 55]. e advantage of using scaffold-free models is determin- can be subdivided into scaffold-free, scaffolded and a hybrid of the two [44, 45]. e air-liquid inter- face (ALI) culture is predominantly used to mimic respiratory tract epithelia in vitro. To establish ALI culture, respiratory epithelial cells are seeded in compartmentalized culture systems on a porous membrane (Transwell® inserts (Sigma-Aldrich, St. Louis, MO, US) or Snapwell™ inserts (Sigma- Aldrich, St. Louis, MO, US)), which physically sep- arates the cells from the underlying media. Cells are allowed to attach and proliferate to form of a con- fluent monolayer, then the culture medium on the apical chamber (Figure 3) is removed. is interface of the cells to the surrounding air forces them to dif- ferentiate and generate a pseudostratified epithelium with different cell populations, while from the basal chamber, the cells have access to the culture medium nutrients and additives via diffusion through the porous membrane [46]. When human nasal or bron- chial or alveolar epithelial cells are grown on an ALI, ciliated and mucus-producing cells differentiate to take on the proper apical-basal morphology, includ- ing functional apical cilia and/or mucous secretion from goblet cells, mimicking the in vivo physiology [43, 47-50]. e ALI culture with respiratory epithelial cells pro- vides a powerful and a physiologically relevant tool to test and develop inhalable products. is is due to ALI culturing, which provides a platform to simulate dissolution in the lung because it can mimic import- ant aspects of the biological milieu in the lung such as pH, clearance mechanisms, mucus and residence time. e lungs are covered with a thin mucus layer, which is a gel lining with varying thickness along the conducting airways down to the alveolar regions. In chronic respiratory diseases, this mucus lining may be dehydrated and thickened, further impairing drug dissolution and absorption. Furthermore, surfactant Figure 3 In vitro cell culture techniques for testing inhaled products Types of Cell Culture 3-Dimensional 2-Dimensional Microfluidic Device Submerged culture: Basal cell monolayer ALI culture: Pseudostratified epithelium Spheroid Organoid Scaffolded Lung-on-a-Chip Apical Basal

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