79 Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research organoid that can develop into branching airways with primitive alveoli [48]. PSC and in particular induced PSC (iPSC)-based organoids have great potential thanks to their potential for combining multiple cell types such as epithelial, smooth muscle cells and fibroblasts [49], but due to their intrinsic immature nature, generating disease models is not straightforward, except for monogenic disorders such as cystic fibrosis (CF) [50]. All in all, lung organoids have rapidly shown their value for respiratory research, thanks to their physiologically relevant properties, the possibility to recreate and understand lung development, and regeneration mechanisms. Organoid amplification [46] and cryopreservation of organoids opens a world of possibilities for the establishment of biobanks to facilitate greater availability of progenitors for organoid development for biomedical research and personalised medicine [51]. LUNG EXTRACELLULAR MATRIX (ECM)-DERIVED HYDROGELS Hydrogels are hydrophilic polymers physically or chemically cross-linked to form a 3D network [52]. Hydrogels can hold copious amounts of fluids while maintaining their structural integrity and represent the hydrated nature of physiological ECM. Individual ECM components or derivatives of them such as collagen, gelatine, fibronectin, fibrin, and hyaluronic acid have frequently been used to form hydrogels to mimic the ECM and provide bioactive components in cell culture [53]. However, they do not fully recapitulate the physiological complexity of the ECM and its macrostructure. Organ-derived ECM recapitulates the tissue-specific biochemical and biophysical complexity of the native tissue [54]. Thus, cell-seeded seeded lung ECM-derived hydrogels have since emerged as an important in vitro system to model the lung microenvironment. The first lung ECM-derived hydrogel was reported by Pouliot et al. in 2016, using porcine lung ECM [55], which was followed by human lung-derived ECM [54, 56] for establishing such models. Lung tissue can be decellularised using different methods, including chemical detergents, freezing/thawing, sonication, enzymatic digestion [53, 57], and recently developed apoptosis-associated approaches [58], followed by lyophilisation and milling into fine power [54] or homogenising using mortar and pestle [53]. The decellularised ECM is solubilised using pepsin in acidic conditions with constant agitation for 24-72 hours [53]. Pepsin preserves the ultrastructure of collagens by cleaving collagen triple helices only at their telopeptide bonds [53], although the digestion time has been shown to affect hydrogel properties and morphology and metabolic activity of cells seeded onto the hydrogels [53, 59]. The pH of the solution is neutralised, followed by buffering with PBS to generate a self-assembling and thermosensitive ECM pre-gel that forms a hydrogel at 37°C [54]. Ultrasonic cavitation has also been 4
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