186 Chapter 8 processes rely on interactions with amine groups of lysine or arginine amino acids, which are known to be parts of cell binding domains including GFOGER, IKVAV or RGD. The implications of methacrylation or thiolation of ECM proteins on cellular functions still need to be explored [16, 18-20]. Alternatively, chemical crosslinking has been applied to ECM-derived hydrogels using harsh chemicals such as glutaraldehyde or genipin, but cytotoxicity limits their use when cells are present in the hydrogel [21]. Another option is using near visible light UV-induced ruthenium/sodium persulfate (SPS) crosslinking, which has been employed on several other types of hydrogels (gelatine or fibrin) with and without cells present in the hydrogels [22, 23]. The higher wavelength (405 nm) of the crosslinking light, which decreases the cytotoxicity, and the lack of requirement for any additional functionalization on the target material are the main advantages of this crosslinking method [24]. Using ruthenium/SPS crosslinking to reinforce the mechanical stability of the ECM-derived hydrogels has recently been reported by Kim et al. [25]; however, the implications of altering the mechanical properties of the hydrogels without changing the (bio)chemical composition have yet to be explored in terms of fibrosis and for developing in vitro models for fibrosis research. In this study, we aimed to develop an in vitro model for examining the influence of mechanical properties of the fibrotic microenvironment by using native lung-derived ECM hydrogels (LdECM), which were generated using ruthenium/SPS crosslinking. We hypothesized that the ruthenium crosslinking would increase the stiffness of lung-derived ECM hydrogels (Ru-LdECM), while the viscoelastic relaxation would decrease, to then trigger pro-fibrotic activation of lung fibroblasts. MATERIALS AND METHODS Porcine Lung Decellularization Porcine lungs (~6-month, female) were purchased from a local slaughterhouse (Kroon Vlees, Groningen, the Netherlands). The lung was dissected, cartilaginous airways and large blood vessels removed, before cutting into ~1cm3 cubes that were homogenized in a kitchen blender prior to decellularization. The lung homogenate was decellularized as previously described [26, 27]. In short, the homogenate was repeatedly washed with Milli-Q® water and centrifuged at 3,000 x g until the supernatant was completely clear. The sedimented material went through two rounds of sequential treatment with 0.1 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), 2 % sodium deoxycholate (Sigma-Aldrich), 1 M NaCl solution and 30 µg/mL DNase (Sigma-Aldrich) in MgSO4 (Sigma-Aldrich) 1.3 mM and CaCl2 (Sigma-Aldrich) 2 mM,
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