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Inhalation February 2023 13 merged culture. In a submerged culture, the cells are grown in a monolayer in media supplemented with growth nutrients. Both primary cells and immortal- ized cell line can be grown using this type of culture technique. It is ideal for initial cytotoxicity studies of products as it enables high-throughput data out- put and testing of broad concentration ranges. Addi- tionally, it can be used to determine if the inhalation products have any pro-/anti-inflammatory and pro-/ anti-oxidant properties properties on the specific cell models being tested. ese tests are normally used as a starting point for "lead" formulation development and optimization in terms of dosing and determi- nation of efficacy and toxicity prior to performance of more specialized and targeted assays using more physiologically relevant 3-D models. Air-liquid interface (ALI) cultures 3-D cell culture models (Figure 3) were developed to recreate the spatial organization of tissue. ey Physiologically relevant in vitro conditions for biological characterization of inhaled products Currently there are several cellular models that are available commercially (Figure 2), that can be used for in vitro testing of inhalation products targeting the respiratory system. ese include immortalized cells lines that can proliferate indefinitely through passaging and primary cells that are freshly isolated directly from donor organs that have a finite life span. ese cells and cell lines can be extracted and established from healthy human tissue or from spe- cific disease states. Figure 2 highlights the models that can currently be purchased from the American Tissue Culture Collection (ATCC) and are representative of the specific regions of the respiratory tract epithelia cells or fibroblasts. ey are color-coded based on the tissue origin, either normal (blue) or diseased (red) subjects. In addition, Table 1 summarizes some ref- erenced examples of their use in inhalation studies. For instance, among the cell lines in red, the CuFi models are derived from a patient with cystic fibrosis and therefore can be used as a model for therapeutic inhalation products targeting cystic fibrosis. Once the appropriate cellular models are selected, there are several cell culture techniques that can be utilized to mimic the physiologically relevant con- ditions for testing of inhalation products. Biological characterization of a product includes tests aimed to conduct a complete assessment of the product when exposed to the respiratory system. ese include test- ing whether the product induces any adverse effects on the respiratory cells, such as cytotoxicity effects of the product (viability assays), inflammation and oxidative stress (addition of inflammatory media- tors or oxidants and adding therapy to look at pro/ anti-inflammatory and pro/antioxidant effects), and effect on tight junction formation (measurement of transepithelial electrical resistance) and permeabil- ity (sodium fluorescein assays) of the epithelial layer. Testing the efficacy of the product is another part of characterization. Functional assays can be used to test the specific mechanism of action of the drug on the relevant respiratory cells. ese include assays that measure downstream effects of the treatment such as enzyme activity (e.g., cAMP assay) or variations in protein expressions. Lastly, cellular models are also particularly useful to determine the mechanisms by which the formulation interacts with the lungs and is absorbed into the systemic circulation by evaluating drug uptake (quantitate intracellular levels), dissolu- tion in the epithelial lining fluid, drug metabolism and transport through the epithelium. Various cell culture techniques have been developed to carry out these standard biological tests of inha- lation products. ey can be sub-categorized into 2-dimensional (2-D) and 3-dimensional (3-D) cell culture (Figure 3). e 2-D category includes sub- Cell Line Name Type References HNEpC Nasal epithelial 6-8 RPMI2650 Nasal epithelial 9-11 Detroit 562 Nasopharyngeal epithelial 12, 13 HuLa-PC Larynx epithelial-like 14 Hs 680.Tr Trachea fibroblast 15 BEAS-2B Bronchial epithelial 16-20 HBEC3-KT Bronchus epithelial 21 NuLi-1 Bronchus epithelial-like 22-26 NL20 and NL20-TA Bronchus epithelial 27-29 HBE4-E6/E7 Bronchus epithelial 19 16HBE14o- Bronchus epithelial 26, 30-32 ChaGo-K-1 Bronchus epithelial 33 NCI-H727 Bronchus epithelial 34 CuFi-1, 4, 5 and 6 Bronchus epithelial 22, 24-26, 35 HBE135-E6E7 Bronchus epithelial 20, 36 BZR Bronchus epithelial 32 A549 Alveolar epithelial 16, 17, 21, 28, 37 SW1573 Alveolar epithelial 38 Calu-1 and 3 Lung epithelial 27, 31, 39-41 NCI-H441 Lung epithelial 42, 43 Table 1 Usage of cell models in inhalation studies