174 Chapter 7 imaging setups are excellent for capturing images in 2D but moving into the third dimension proves challenging to visualise. Lung ECM-derived hydrogels are not translucent, like many of the single ECM or synthetic hydrogels, and this opacity challenges the optical depth of field. The autofluorescence of the lung ECM generates a very noisy image when using many traditional fluorescent reporters. Finally, tracking cell behaviours over time in 3D is extremely difficult to automate when the cells continuously move out of the plane of focus. Advances in imaging and capturing information from cells when they are interacting within their microenvironment is urgently needed to facilitate the full capacity of lung ECM-derived hydrogels. DISCUSSION AND FUTURE REMARKS: Studies in lung ECM-derived hydrogels will help to inform us of the optimal microenvironment for different cell types, as the cells continuously remodel their environment in, what appears to be, a programmed response. Whether there is temporal regulation of the remodelling, in particular in response to injury, is an outstanding question for the field. How these processes are altered in chronic lung diseases, and whether the progression of such processes can be reversed is knowledge that can be informed through the use of lung ECM-derived hydrogels. The approaches described above, including modulating mechanical properties of ECM-derived hydrogels without changing the ECM composition and the application of mechanical forces to cells within a 3D microenvironment, are attractive as they will facilitate research enabling the field to begin separating influences of the mechanical changes from those of the biochemical changes in the ECM in lung diseases. Such elucidation may open the door for development of mechanosensitive therapeutic targets for lung diseases. To fully leverage the advantages offered by lung ECM-derived hydrogels multidisciplinary teams who bring together expertise from the diverse fields needed to advance such systems will be necessary (Figure 2). Innovative researchers from pulmonology, cell and molecular biology, polymer chemistry, biomedical engineering, imaging and physics backgrounds are all needed to maximise opportunities and ensure the current challenges quickly become advantages for this exciting, emerging area of lung disease research.
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