137 3D lung models – 3D extracellular matrix models When culturing cells in a 3D environment using models such as hydrogels or matrices, the cells have the opportunity to adhere in all dimensions to the matrix fibers that surround them, and consequently do not experience forced polarity [77, 80, 81]. These engagements occur via the binding of cell surface integrins to defined motifs (e.g., arginine-glycine-aspartate (RGD) motifs on fibronectin)) present on ECM proteins. Through this binding, cells sense the stiffness of the surrounding ECM, a process referred to as mechanosensing [82-86]. The mechanical stimuli from the ECM are then converted into biochemical activity activating intracellular signaling pathways resulting in gene expression responsible for regulating cell survival, proliferation, differentiation, apoptosis, ECM protein synthesis and secretion [85]. In addition, matrix fibers sequester soluble factors (e.g. growth factors, cytokines, extracellular vesicles, chemokines) and nutrients through entrapment and binding to proteoglycans and GAGs, potentially exposing cells to a gradient of nutrients, growth factors and soluble factors [77, 79, 87, 88]. The addition of a third dimension is a logical step forward to make the models used for studying cell interactions in lung diseases more translational, and with the incorporation of the ECM these innovative models will allow the creation of more intricate co-culture systems to answer more complex questions about lung pathophysiology [89]. 3D MODELS – 1: SINGLE-PROTEIN MODELS Collagen One of the most commonly used 3D models which consist of one ECM protein are models based on collagen type I. Collagen type I is commercially available, but it can also easily be derived from rodent, porcine, bovine or human sources, and can be relatively cheap to produce in large quantities [90]. Secondly, collagen can be used in different forms of biomaterials such as hydrogels or sponges [91]. Since collagen is the most abundant ECM molecule in native tissues, which modulates and supports the survival of different cell types, it makes collagen scaffolds a good model for mimicking in vivo tissue. Cells can be cultured in 3D collagen models for days and weeks. Cells grown in such an environment are likely to maintain similar behavior, migration, attachment through GFOGER, GVOGEA, GLOGEN and other sites and signaling pathways to those enacted in the lungs [92, 93] (Figure 2). Recently, 3D collagen scaffolds have been used for exploring alveolar recovery and angiogenesis after lung injury. After the implantation procedure, a collagen scaffold lost 30% of its size by the 14th day and had almost completely degraded by the 90th day in vivo [94]. In other research, a 3D collagen model has been used to deliver epithelial cells and fibroblasts to rabbit trachea in vivo [95]. Thirdly, the ability to modify gel stiffness 6
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