236 Chapter 9 Lyophilization and grinding of decellularized lung samples Decellularized lung samples were lyophilized in order to remove excess water. Briefly, the samples were snap-frozen using liquid nitrogen, then freeze-dried using a Labconco Freezone 2.5 Liter Benchtop Lyophilizer (Labconco, Kansas City, MO, USA) until the samples reached complete dryness. Once the lung scaffold samples were dry, they were brought to room temperature for the grinding process. An IKA A11 Basic Analytical Mill (IKA, Germany) was used to grind the lung scaffold pieces to a fine powder. Lung ECM powders were kept at room temperature with desiccant until use. Figure 6: Overview of the experimental design and readouts measured in this study. A) Control and IPF lungs were decellularized, ground to a fine powder and digested using pepsin in acidic medium. The solubilized lung ECM was used to prepare lung ECM-derived hydrogels. B) Combinatorial approach used in the experiments. Fibroblasts and ECM groups were cross-combined to have combinations of control and IPF originated samples in every experimental batch. C) The readout applied in this study: PicroSirius Red and Alcian Blue stained sections of empty and fibroblast-seeded control and IPF lung ECM-derived hydrogels were scanned and digitally analyzed for area only or area and intensity (Characterization Readout #1). Fluorescence images of PicroSirius Red stained sections of empty and fibroblast-seeded control and IPF lung ECM-derived hydrogels were analyzed using TWOMBLI plugin in ImageJ to analyze the fiber characteristics (Characterization Readout #2). The mechanical properties (stiffness and stress relaxation) of empty and fibroblast-seeded control and IPF lung ECM-derived hydrogels were measured using low load compression tester (Characterization Readout #3).
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