Micropatterned surfaces to engineer focal adhesions for analysis of cell adhesion strengthening

Cell adhesion to extracellular matrices is critical to numerous biomedical and biotechnological applications. Cell adhesion involves integrin receptor-ligand binding and adhesion strengthening, which includes integrin clustering, interactions with cytoskeletal and signaling components to form specialized focal adhesion structures, and cell spreading. Because of the complex nature of the strengthening process, quantitative analyses of cell adhesion have been limited to initial events. In this study, we applied micropatterning methods to control focal adhesion size and position, and decouple integrin clustering and focal adhesion assembly from gross changes in cell morphology. This strategy allowed analysis of the evolution of adhesion strength independently of cell spreading and redistribution of adhesive structures. Microcontact printing was used to pattern alkanethiol self-assembled monolayers into arrays of circular adhesive islands (2, 5, and 10 mum diameter) within a nonadhesive background. Fibroblasts adhered to fibronectin-coated islands and remained nearly spherical. The cell-substrate contact area was constrained to the micropatterned domain and immunofluorescence staining revealed robust assembly of adhesive structures containing components associated with conventional focal adhesions. Cell adhesion strength to fibronectin-coated micropatterned islands was quantified using a spinning disk device that applies a well-defined range of hydrodynamic forces to adherent cells. Adhesion strength exhibited significant time- and adhesive area-dependent increases. Comparison of experiments for similar contact areas at different time points showed a 9-fold increase in adhesion strength over time, independently of cell spreading. These results demonstrate that focal adhesion assembly, independently of changes in cell morphology and redistribution of adhesive structures, contributes significantly to adhesion strengthening. This work provides an experimental framework for the functional analysis of focal adhesion structural and signaling components in physiological and pathological conditions.