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The mechanical properties of the extracellular microenvironment regulate cell behavior, including migration, proliferation and morphogenesis. Although the elastic moduli of synthetic materials have been studied, little is known about the properties of naturally produced extracellular matrix. Here we have utilized atomic force microscopy to characterize the microelastic properties of decellularized cell-derived matrix from human pulmonary fibroblasts. This heterogeneous three-dimensional matrix had an average thickness of 5±0.4μm and a Young’s modulus of 105±14Pa. Ascorbate treatment of the lung fibroblasts prior to extraction produced a twofold increase in collagen I content, but did not affect the stiffness of the matrices compared with matrices produced in standard medium. However, fibroblast-derived matrices that were cross-linked with glutaraldehyde demonstrated a 67% increase in stiffness. This work …