This paper presents a readily accessible patterningplatform-based upon geometric constraints, discrete cell suspension droplets, and controlled cell settling-that provides both temporal and spatial patterning capabilities. As a demonstration, single-cell (and bead) suspensions as well as multicellular human embryonic stem cell colonies are spatiotemporally patterned onto arbitrary substrates. These substrates include tissue culture surfaces, cell monolayers, protein-coated surfaces, and 3D gel matrices. The generation of soluble factor gradients is also demonstrated. This method is completely passive and does not require external power sources. Spatiotem-poral patterning provides a foundation for future biological studies that explore the time-dependent relationships between cell-cell signaling and cellular responses.
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