## 3 Lower Respiratory-Physiology-On-A-Chip
### 3D Lower Respiratory-Physiology-On-A-Chip
#### 3.2.1 Transwell-Based Air-Liquid Interface Respiratory Model  
[PERSON] et al. [48] compared and evaluated the similarity to the in vivo airway epithelium when human airway epithelial cells (Calu-3) and primary human airway epithelial cells (hAEC) were immersed at the ALI and a plastic surface, respectively. This study found that ALI cultures with the use of primary cells have similar transcription profiles to epithelium in vivo. Therefore, transwell-based 3D cultures have been used recently as a standard culture protocol to characterize the newly developed experimental cell sources. [PERSON] et al. [49] evaluated the characteristics of primary normal human bronchial epithelial (NHBE) cells using transwell-based ALI culture conditions to understand the effect of culture conditions on cell growth, epithelial phenotypes, and function. Primary NHBE cells were cultured for up to 6-8 passages in the ALI environment, and it was confirmed that bronchial epithelial phenotype, cell integrity, differentiation, and ion channel function were well maintained. In addition to primary hAEC, many groups used mouse organ epithelial cells (MTECs). However, MTECs has the disadvantage of requiring a large number of mice for cell isolation since it is difficult to grow in vitro. Thus, [PERSON] et al. [50] developed ALI culture conditions and a new combination of medium components to maintain the differentiation capacity and proliferation of MTECs. Recent studies have also demonstrated that ALI culture conditions are important factors for the directed differentiation of induced pluripotent stem cells into airway and alveolar epithelial cells [51; 52]. Primary hAEC ALI culture provides a platform for investigating the mechanism of differentiation and dynamic recovery process after airway epithelial injury, as well as a method for studying a fully epithelialized epithelial layer.  
In vitro toxicity studies of nanoparticles (NPs) that enter the respiratory tract and accumulate in the lungs are typically performed under immersion conditions, in which NPs are suspended in the cell culture medium; therefore, it is difficult to determine the adverse effect of NPs on the accumulation in cells appropriately. Moreover, NPs easily changed their morphology (e.g., precipitation and aggregation) under immersion condition. The cellular response to NPs under immersion culture conditions is completely distinct from those observed in the ALI environment. To mimic the actual pathological features associated with inhalation, [PERSON] et al. [53] exposed human lung epithelial cells (A549) to aerosols (silica NP) at the ALI culture conditions. They found a lower cellular sensitivity to silica NPs under ALI culture conditions for cytotoxic and inflammatory responses compared to the conventional immersion culture results.  
Although it is well-known that cigarette smoke (CS) affects the innate immune function of airway epithelial cells and epithelial repair, direct assessments of the impact of CS have not yet been fully understood. Most studies applied an aqueous extract of CS to an undifferentiated primary airway epithelial cells, which is difficult to reflect local conditions of the smoker's lungs. [PERSON] et al. [54] exposed CS to an ALI culture model of primary bronchial epithelial cells (ALI-PBEC) to investigate the effects of CS on airway epithelial repair. The results suggested that CS adversely affect the immune response for repair of damaged airway epithelial cells and contribute to the development and progression of COPD. Thus, the ALI respiratory model has been used in numerous respiratory studies for its ability to more closely simulate the actual in vivo environment compared to the 2D or 3D immersion models. However, limitations remain in simulating the actual respiratory environment that facilitates gas exchange and nutrient supply through the blood flow.