Patterning cells in highly deformable microstructures: Effect of plastic deformation of substrate on cellular phenotype and gene expression

We describe the fabrication of deformable microstructures by low-pressure—soft—microembossing (μSEmb) that provides in vitro experimental “test-beds” to investigate the interplay of mechanical and chemical stimuli on cell behavior in a highly controlled environment. Soft microembossing exploits the softness and plasticity of parafilm to fabricate microstructures by pressing a silicon master or an elastomeric poly(dimethylsiloxane) stamp into the parafilm. We demonstrate that a protein-resistant comb polymer can be printed into the raised features of the embossed microstructures, which imparts protein, and hence cell resistance to those regions of the microstructures. These two features of our fabrication methodology—microembossing followed by spatially selective transfer of a nonfouling polymer—forms the core of our strategy to pattern cells within the parafilm microstructures, such that the cells are confined within bottoms of the microstructures. Cell culture experiments demonstrated the preferential cell attachment of NIH 3T3 fibroblasts to the fibronectin (FN) micropatterns by immunofluorescence microscopy. The actin cytoskeleton realigned along the axis of applied mechanical stress, and stretched cells showed altered gene expression of cytoskeletal and matrix proteins in response to mechanical deformation. The use of parafilm as a substrate and μSEmb as a fabrication method provides a simple and widely accessible methodology to investigate cellular behavior under well-defined conditions of plastic deformation and surface ligand density.