Cell migration is controlled by the integration of numerous distinct components. Consequently, the analysis of cell migration is advancing towards comprehensive, multifaceted in vitro models. To accurately evaluate the contribution of an underlying substrate to cell motility in complex cellular environments we developed a migration assay using magnetically attachable stencils (MAts). When attached to a culture surface, MAts create a defined void in the cell monolayer without disrupting the cells or damaging the underlying substrate. Quantitative analysis of migration into this void reveals the substrate's contribution to migration. The magnetically-guided placement of a microfabricated stencil allows for full experimental control of the substrate on which migration is analyzed. MAts enable the evaluation of intact, defined matrix, and make it possible to analyze migration on unique surfaces such as micropatterned proteins, nano-textured surfaces, and pliable hydrogels. These studies also revealed that mechanical disruption, including the damage that occurs during scratch assays, diminishes migration and confounds the analysis of individual cell behavior. Analysis of migration on increasingly complex biomaterials reveals that the contribution of the underlying matrix depends not only on its molecular composition but also its organization and the context in which it is presented.
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