90 Chapter 4 Isotope-labelled amino acids that tag chemical, metabolic, and enzymatic processes have been used to differentiate, in combination with mass spectrometry, between de novo ECM synthesis and native ECM proteins in decellularised PCLS [26, 152]. While mass spectrometry analysis on regional and disease-specific empty lung ECM hydrogels has been recently performed [153], employing such analysis on cell-seeded lung ECM-derived hydrogels is yet to be completed. Parallel to mass spectrometry, Raman spectroscopy is a label-free method that allows investigation of biochemical composition of ECM based models such as PCLS, decellularised PCLS, and hydrogels [154]. Regarding gene level characterization, RNA-sequencing approaches have been successfully applied to PCLS [155], organoid [46], and LOC systems [156]. Single-cell RNA-sequencing, a method based on capturing the mRNA content of each cell at a single cell resolution has been recently applied to lung organoid samples [157]; however, it has yet to be implemented on the remaining systems. Metabolomics approaches have also been applied to PCLS [146] and iPSC-derived epithelial progenitor cells [158]; however, no application of these approaches to other model systems has been reported. Mechanical Characterization Lung diseases are often characterised by an alteration in physiological mechanical properties of the ECM, and thus of the lung tissue as a whole. These changes in turn affect cellular behaviour. Importantly, the macroscopic stiffness of a tissue is rarely the same as the local microscopic stiffness which is sensed by cells through their cell surface focal adhesions [159]. Mechanical properties of in vitro models (mainly hydrogel based) can be manipulated to mimic disease conditions and characterised using various methods including rheometry [85], low-load compression testing [54, 68, 160] and atomic force microscopy (AFM) [61]. On a cellular, and thus a micrometrescale, spatial elastic modulus can be measured using Brillouin microscopy. Brillouin light scattering allows for live measurements of viscoelastic properties over the surface of the sample and can be applied on bioprinted cells and 3D in vitro models [161, 162]. Innovative use of 4D traction force microscopy and an open source MATLAB software package represents an emerging method to measure and calculate traction forces exerted by cells in 3D hydrogel cultures over time [163]. On a microscopic scale, AFM has been commonly used to both measure stiffness and viscoelastic properties which allows for a local assessment of the material properties [164]. Cells in the lungs experience dynamic forces and are under constant cyclic stretching, which is exaggerated in patients being mechanically ventilated [165]. Additionally, cells also receive biomechanical cues from the local microenvironment [166, 167]. A novel approach using AFM was highlighted as a way to measure microrheology of tissue exposed to different levels of stretch [168].
RkJQdWJsaXNoZXIy MTk4NDMw