145 3D lung models – 3D extracellular matrix models elsewhere [140]. Therefore, the use of decellularized tissue for 3D modeling is an advantageous technique in terms of mimicking native tissue structure and composition. However, these models are still a long way from modeling the complexity of lung tissue. The development of long term functional units for gas exchange is of utmost importance. However, this requires both- epithelized airways and endothelialized vessels. Vascularization and innervation of decellularized tissue, seeding, and expanding multiple cell types and lineages together, and developing appropriate methods to measure experimental outcomes are some of the major challenges yet to be overcome. Decellularized lung ECM-derived hydrogels Although single protein hydrogels can mimic the mechanical properties and elevate the cell culture model to 3D, they do not represent the full complexity of the matrisome. Next to using decellularized matrices, another approach for incorporating the complexity of the ECM into 3D models has emerged in recent years, lung-derived ECM hydrogels [58, 163]. These hydrogels are generated from solubilized decellularized lung ECM (Figure 3B). The decellularized ECM is lyophilized and milled into a fine powder, to increase the surface area of the ECM to aid in the solubilization process. The solubilization of the ECM has most often been performed via pepsin digestion in an acidic buffer [58, 163, 164]. During pepsin digestion, the ECM proteins are enzymatically solubilized into a monomeric suspension, generally under constant agitation for an extended time (24-72h, although this varies for different tissues) at room temperature [164]. After digestion, the pH of the solution is neutralized and buffered with PBS to prepare a thermosensitive ECM pre-gel solution which spontaneously self-assembles into a hydrogel when incubated at 37°C [165]. Recently, it has been shown that ultrasonic cavitation could also be used to solubilize the milled ECM, although the source of ECM was not lung tissue [166]. The whole process disrupts the original complex ultrastructure of the starting tissue ECM and reduces it to a suspension of its multitude of components. The pepsin solubilization process needs to be tailored to the specific organ and the success is dependent upon the pepsin digestion time which affects the mechanical properties such as stiffness and dictates the subsequent effect of the ECM hydrogel on cells [167]. Another important mechanical property for hydrogels is viscoelasticity. Viscoelasticity describes how a material that has both viscous (water, soluble factors) and elastic (ECM proteins) components, distributes forces when a stress is applied [168]. Both stiffness and viscoelasticity have been found to influence cell behavior such as spreading, proliferation and differentiation [169, 170]. For the lung, hydrogel models that incorporate the entirety of the ECM have been made from porcine lungs [163, 171, 172] and human lungs [58]. For now, most cell experiments using 6
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