148 Chapter 6 scaffold in its native form brings the opportunity of utilizing the complex architecture of lung tissue in in vitro studies; however, introducing cells back to these samples is not challenge-free. Different strategies of decellularization generate differences between ECM components, structure, and mechanical properties of the obtained scaffold. The impact of these differences on recellularization remains unclear. Ensuring an appropriate 3D distribution of the cells with their correct physiological placement remains a major limitation in using decellularized lung pieces. Additionally, factors such as cell source and seeding density, optimal medium composition, flow rate, and accessibility of the injection site also play a key role in determining the success of recellularization [175]. Long term storage (> 1 year) of decellularized tissue has also proven challenging due to the loss of ECM structure and reduced mechanical and angiogenic properties [176]. Lung ECM-derived hydrogels, on the other hand, can address this problem by providing the control over shape and construction but lack the topographical organization of the ECM present in vivo. Since cells can be spatially introduced to the different parts of the hydrogel(s), placing the cells in a physiologically representative manner is possible when using these hydrogels. However, the current methodology to prepare such hydrogels is rather limited: pepsin digestion is currently the most applied method [164]. While the mechanical properties of these hydrogels can resemble the lung tissue in health and diseased states, the implications of the digestion procedure have recently been discussed [58, 167]. Another limitation in the ECM-derived hydrogels for creating 3D models for in vitro research is the mechanical tunability. Mechanical properties can be changed by varying the concentration of the initial ECM input, but this variation also changes other properties such as pore size and cell binding site availability. Recent studies have focused on chemically modifying the ECM-hydrogel solutions to introduce a chemical crosslinking for the possibility of tuning mechanical properties [172], while more research is required to understand the potential implications of these modifications other than changing mechanical properties. CONCLUSIONS In vitro models for mimicking the lung microenvironment have advanced greatly, especially in the last two decades. While conventional 2D cell and tissue culture models are being routinely used, there is an increasing trend towards research performed using advanced models. One of the most important characteristics of these advanced models is the improved dimensionality. A 3D culture environment
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