135 3D lung models – 3D extracellular matrix models applied for the study of mechanisms underlying lung diseases [69]. However, several limitations using 2D models have been identified when trying to replicate the 3D in vivo environment including differences in cell – cell and cell – ECM interactions, as summarized in Table 1. Other limitations of 2D models include adhesion of cells only in a 2D plane and induction of apical – basal polarity of the cells which can influence apoptosis signaling pathways [67, 70]. The stiffness of basic 2D culture surfaces, such as tissue culture plastic (TCP), is significantly higher than the in vivo microenvironment, resembling cartilage or bone tissue rather than soft lung tissue. Even in fibrotic diseases such as IPF, the increased Young’s modulus is not as high as TCP which is 1 GPA compared to 16 kPa, respectively [49, 71]. Increased substrate stiffness leads to greater proliferation of cells and differentiation of fibroblasts to myofibroblasts [72, 73]. The highlighted differences between 2D and 3D culture models affect cell behavior, which contributes to cellular alterations that affect phenotype, differentiation, proliferation and gene and protein expression, cell signaling and behaviors [67, 68, 73]. Classical 2D models provide limited opportunities for studying cell migration and tissue remodeling; with perhaps the exception of information that can be garnered from “wound healing” experiments. However, in recent years, novel 2D models that feature properties that further reflect those of lung tissue have been developed. For example, to study the effects of mechanical properties on cell behaviors mechanically tunable polydimethylsiloxane substrates and polyacrylamide (PA) hydrogels can be used. Moreover, ECM proteins can be covalently cross-linked to the PA hydrogels [74]. The growth of cells on a basement membrane extract, such as Matrigel, provides signaling engagement, cell integrity and structural support which are unattainable in the basic 2D models [75]. Another semi 2D model that has been adopted is an air-liquid interface (ALI) culture system, where epithelial cells are grown on an upper surface of a porous membrane. The apical side of the membrane can be used to expose cells to air. The basal side is maintained in contact with the culture media to provide a continuing supply of nutrients. This model is commonly used for the culture of bronchial epithelial cells, which differentiae into pseudostratified mucociliated epithelial cells after exposure to air, to test the influence of external stimuli on these cells [76]. 6
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