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16 February 2023 Inhalation ACI stage plate is 3-D printed to enable housing of Snapwell inserts. Similarly in Figure 4B, a printed, modified NGI stage plate to house Snapwell inserts is used to replace the NGI stage. ese modified plates can be replaced with an ACI or NGI plate at any of the impactor stages that represent the trachea through the alveoli, depending on the target for the inhalation product, and can be used to evaluate the differential interactions of aerosols with the various regions of the respiratory tract. After the aerosolized dose has been delivered, the Snapwell inserts contain- ing the epithelia can be removed and tests for biolog- ical characterization can be conducted [4, 9, 66-68]. Similarly, the pharmacopeia-approved nasal expan- sion chamber used to assess delivery of nasal spray formulations can also be 3-D printed to include housing for inserts [9]. Co-cultures Co-culture systems are used to study the fundamen- tal interactions and behaviors between different cell populations. Co-cultures are cell cultivation set- ups in which two or more different populations of cells are grown with some degree of contact between them. is enables the study of natural interactions between populations and generates a microenvi- ronment (involving cytokines, growth factors and transcriptional regulators) that reflects in vivo-like human tissue and can be much more relevant than animal models when performing drug studies [69]. e most popular types of co-culture in respiratory research are bi-cultures and triple-cultures (Table 2). Co-cultures of airway epithelial cells, macrophages, dendritic cells and fibroblasts are instrumental in dis- ease modeling, drug discovery and development of novel therapeutic targets [70]. ever, such testing is significantly different than pro- cesses that occur after inhalation of "real" airborne aerosols or involve the complex geometry and respi- ratory mechanics of the lungs. An alternative model with which to study aerosolized inhalation products is the integration of ALI culture models with the pharmacopeia-approved impaction models used to assess lung delivery and nasal expansion chambers to assess intranasal delivery. For lung delivery, inertial impaction studies are con- sidered the gold standard for in vitro assessment of the aerodynamic deposition of inhaled formulations. Such tests are regulatory requirements to estimate the amount of formulation that is delivered to the lungs [63]. Impactors can classify particles according to the aerodynamic diameter of the entire delivered dose [64] and quantitate the delivered amount of the formulation at the various stage of the human respi- ratory system. e type of impactor to use for anal- ysis depends on the inhalation product tested and can be used on dry powder inhalers (DPIs), nebuliz- ers, pressurized metered dose inhalers (pMDIs) and nasal sprays [65]. e impactors alone, however, can- not determine efficacy, drug uptake, dissolution and transport through the epithelium. To address these parameters, hybrid approaches and modifications have been developed that involve combining the bio- logical representation of the lung epithelium (ALI models) with the impaction instrumentation. ALI cultured epithelial models generated from cell/ cell lines representing the various regions of the lung can be inserted into the respective stages of an impac- tor to generate a model that not only can assess the regional lung deposition of an aerosolized formula- tion but also its biological impact. Figure 4 illustrates such an integrated model using the Andersen cascade impactor (ACI) in Figure 4A and the Next Genera- tion Impactor (NGI) in Figure 4B. In Figure 4A, an Type Description Examples Bi-Culture • Uses two cell types • Can be used with submerged culture, ALI, spheroids, organoids and scaffolded culture models • Can be mixed or separated by membrane • A549 cells and fibroblasts spheroids to test anti-cancer nanoparticle delivery [71] • Human bronchial epithelial cells and airway smooth muscle cells to test inflammatory modulation of salbutamol and budesonide [72] • Calu-3 cells grown with bacterial biofilm (P. aeruginosa) to test the efficacy of antibiotic treatment with ciprofloxacin [73] Triple-Culture • Uses three cell types • Generally used with chambered culture with ALI, and spheroid and organoids • Usually, two cell types mixed or seeded with one in a different chamber • A549 or 16HBE14o- epithelial cells co-cultured with macrophages and dendritic cells to study interaction with xenobiotics [74] • Human pulmonary microvascular endothelial cells co-cultured with NCI-H441 cells and differentiated blood monocytes (THP-1) to study inflammation and interaction of nanocarriers [75] • A549 cells co-cultured with macrophages and dendritic cells to test the effects of allergens [76] Table 2 Co-culture types used for inhalation studies