Microstencil-based spatial immobilization of individual cells for single cell analysis

Cells exhibit biologically heterogeneous phenotypes, particularly in pathogenic states. To study cell behavior at the single cell level, a variety of micropatterning techniques have been proposed that allow the spatial organization of cells with great control over cell volume, morphology, and intercellular interactions. Among these strategies, microstencil patterning has traditionally been eschewed due to fragility of membranes and lack of control over cell configurations within patterns. Here, we present a simple and reproducible strategy to create robust microstencils and achieve consistent and efficient cell patterns requiring less than 4 μl of cell solution. Polydimethylsiloxane microstencils fabricated with this technique can be used dozens of times over the course of several months with minimal wear or degradation. Characterization of pattern size, cell suspension density, and droplet volume allows on-demand configurations of singlets, doublets, triplets, or multiple cells per individual space. In addition, a novel technique to suppress evaporative convection provides precise and repeatable results, with a twofold increase in patterning efficacy. Selective dual surface modification to create hydrophilic islands on a hydrophobic substrate facilitates a significantly longer and healthier lifespan of cells without crossover of pattern boundaries. The ability to pattern individual cells with or without an extracellular matrix substrate and to control the magnitude of cell-cell contact as well as spread area provides a powerful approach to monitoring cell functions such as proliferation and intercellular signaling.