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Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel\u2010bundled long fibrils by changing collagen polymerization temperature. This permits analysis of 3D cell migration in different ECM architectures found in vivo while maintaining a similar ECM concentration. 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