235 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment In summary, we examined how a 3D fibrotic microenvironment is interacting with fibroblast remodeling of this environment by both control and IPF fibroblasts using native lung ECM-derived hydrogels and primary lung fibroblasts. Through employing native ECM from control and IPF lungs, most biochemical and biomechanical properties of control and IPF lungs were mimicked in these hydrogels, thereby presenting an improved model system for investigating the interplay between the microenvironment and fibroblasts during the fibrotic process. Considering the lack of physiologically replicative models available for basic and translational research on generating treatment strategies targeting fibrosis, employing in vitro models derived from human-sourced materials can pave the way towards better understanding of fibrosis and better drug discovery processes. MATERIALS AND METHODS Experimental Design The experimental approach adopted in this study is described in Figure 6. The specific details of the methods are described below. Lung decellularization Decellularized control (macroscopically normal tissue, referred to as control throughout the manuscript) and lung tissue from patients with IPF were kindly provided by Dr. Steven Huang, University of Michigan, USA. De-identified control and IPF human lung tissue were provided by the University of Michigan; as the tissues were de-identified and coming from deceased donors, the University of Michigan Institutional Review Board deemed this work exempt from oversight. The decellularization procedure was performed as described previously [11]. Briefly, the fresh lung tissue samples were washed with 1X PBS (Gibco, Waltham, MA, United States). The tissue sample was washed for 24 hours per step at 4°C (unless otherwise stated) with the series of different solutions under constant agitation conditions: 1% (v/v) Triton X-100 (Sigma Aldrich, St. Louis, MO, USA), 2% (wt/v) sodium deoxycholate (Sigma Aldrich), 1 M NaCl (Sigma Aldrich), 30 mg/L DNase (Sigma Aldrich) with 1.3 mM MgSO4.7H2O (Sigma Aldrich) and 2 mM CaCl2 (Sigma Aldrich) at 37°C. The washing series was performed twice with three times PBS washes between every different solution. Afterwards, the ECM samples were treated with 0.18 % peracetic acid solution (32% w/w; Merck, Darmstadt, Germany) in 4.8% ethanol (Fresenius Kabi, Bad Homburg vor der Höhe, Germany) solution for 24 hours at 4°C with constant shaking. Lastly, the ECM samples were washed using PBS and kept in PBS (+1% Penicillin-Streptomycin (Gibco)) at 4°C until the next step. 9
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