294 Chapter 10 Another intriguing application of my method described in Chapter 8 would be using it in organoid culture systems, such as organoids derived from IPF lungs (as described in Chapter 5) to investigate how stiffer ECM influences the interplay between epithelial cells and supporting cell populations. Investigating the cross-section of fibrotic mechanics and fibrotic ECM-imprinted cells in the context of alveolar epithelium regeneration would reveal additional dynamics and interactions that the current limit of our knowledge has not even reached. Another implication of the findings in my thesis translates to organoid culture using mesenchymal cells embedded in Matrigel; the potential pathological instructions delivered from Matrigel, being derived from sarcoma ECM [33], towards fibroblasts may have consequences for how these fibroblasts remodel the environment and hence signal to epithelial cells. Examining the interaction between biomechanics of fibrotic ECM and resident cells during lung fibrosis in in vitro models with multiple cells types will advance our understanding on the perpetuation of the fibrotic response. The opportunities provided by 3D in vitro models can be further strengthened by using materials of human origin. One of the initial reports using such material was by Booth et al., in which they reported the use of decellularized ECM derived from fibrotic human lungs as an ideal system to be used in in vitro culture [13]. In the same study, they have also observed fibrotic ECM-triggered changes in lung fibroblasts [13]. Our understanding of cell-matrix interactions in fibrosis was furthered by Parker et al., who demonstrated a positive pro-fibrotic feedback loop between fibrotic ECM and fibroblasts [34]. While these pioneering studies have advanced the possibilities of recapitulating the lung microenvironment in vitro, the challenges in recellularization of intact decellularized matrices with respect to size and shape of the matrices remained as a roadblock [35-37]. Lung ECM-derived hydrogels provide a unique opportunity to bypass the limitations of intact matrices in size and shape, as well as facilitating the (re)introduction of cells (as discussed in Chapter 7). Based on this, in Chapter 9, I have mimicked fibroblast-ECM interactions in the context of IPF by comparing the responses of primary lung fibroblasts isolated from control and IPF lungs and seeded in either IPF or control lung ECM-derived hydrogels. I characterized these fibroblast-seeded ECM-derived hydrogels with respect to collagen and GAG content, collagen fiber organization and mechanical properties, and compared them to their empty counterparts. I showed that control fibroblasts and IPF fibroblasts responded differently to fibrotic microenvironment: for most parameters, the responses of control fibroblasts in fibrotic microenvironment were towards making the environment even more fibrotic as shown by decreased high-density matrix, increased fiber curvature, increased stiffness of the hydrogels, compared to the empty hydrogels, while IPF-derived fibroblasts exerted less pronounced effects.
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