Direct Micropatterning of Extracellular Matrix Proteins on Functionalized Polyacrylamide Hydrogels Shows Geometric Regulation of Cell-Cell Junctions

Microcontact printing of extracellular matrix (ECM) proteins in defined regions of a substrate allows spatial control over cell attachment and enables the study of cellular response to irregular ECM geometries. Over the past decade, numerous micropatterning techniques have emerged that conjugate ECM proteins on hydrogel substrates of tunable stiffness, which have revealed a range of cellular responses to varying matrix stiffness and geometry. However, micropatterning of ECM proteins on polyacrylamide (PA) hydrogel remains inconsistent due to its unreliable conjugation with the commonly used protein cross-linkers, particularly at low stiffness. To address these problems, we developed a micropatterning technique in which the PA gel is functionalized by incorporating oxidized N-hydroxyethylacrylamide, which allows direct protein binding through reactive aldehyde groups without any exogenous cross-linkers. As a result, a uniform and consistent protein transfer onto the hydrogel substrates of defined geometries is achieved, even for soft PA gels. We formed square, rectangular, and triangular patterns of two constant areas on soft and stiff PA gels that provide large and small adhesive areas for the MCF10A human mammary epithelial cell pairs. We measured intercellular E-cadherin (E-cad) expression and found that cell-cell junctions could be deteriorated independently by either the stiff ECM of any shape or the elongated cell morphology, accompanied by increased cell-generated tractions, on rectangular soft ECM patterns. Inhibition of nonmuscle myosin II reduced the E-cad junctional localization in cell pairs. When the cell spreading was restricted by reducing the adhesive area of the patterns, we observed an overall rise in E-cad expression at cell-cell junctions. Our findings present an improved micropatterning technique which reveals a geometric regulation of cell-cell junctions in epithelial cell pairs.