89 Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research CHARACTERISATION OF IN VITRO MODELS Today the use of complex 3D systems has made it possible to undertake translational studies in models that more closely mimic the in vivo situation. State-of-theart characterisation of these 3D in vitro models is thereby of importance. The characterisation of experimental 3D models such as PCLS, organoids, (cell-seeded) ECM-derived hydrogels and decellularised lung matrices, and LOC, presents different challenges, with appropriate ways for characterising biological and physical aspects within the models dependent on the research question being addressed. These research questions might include different conditions, including but not limited to basal levels, challenged and stimulated. Here we present a non-exhaustive summary of recent advances in characterization of these in vitro models. Imaging Imaging is a major methodology used for characterisation of the macroscopic architecture and spatial localisation of specific proteins or cells, as well as cellcell and cell-ECM interactions [142]. Light sheet fluorescence microscopy (LSFM) is an emerging technique for imaging larger 3D samples with high spatiotemporal resolution [143]. LSFM has been used extensively for organoid development studies, where visualisation is essential for understanding the cellular complexity [144] and airway development [145]. LSFM, however, requires fixation of the sample and optical clearing which is a challenge when working with 3D structures with different thickness such as PCLS. This is due to the difficulty with optical clearing in thicker PCLS that render blurry images at ≥100 μm depth. Scanning electron microscopy (SEM) has also been used to characterize cellular structures in PCLS [146], lung organoids [46, 147], and LOC [108]; or fibre structure of empty lung ECM-derived hydrogels [56]. Second harmonic generation microscopy [148, 149] has greatly facilitated the characterisation of ECM components in a PCLS model [146]; however, it has yet to be applied to other model systems for characterization of cells and microenvironment. The imaging technique employed depends on several properties of the particular in vitro model, thus limiting the applicability of different microscopic approaches to certain 3D models. Some important considerations while choosing an imaging technique for an in vitro model include: the thickness and size of the sample, effect on live cells, and fixation methods. Protein and Gene Level Characterizations Recent advances in sensitivity and resolution of mass spectrometry allow for deeper characterisation of all 3D models [26, 150] and have been commonly used for the characterisation of ECM composition, ECM remodelling, and cell phenotyping [151]. 4
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