Sequential stenciling to reconstitute 2D microtissues for multicellular and synthetic signaling architectures.

In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2Dinvivoplatforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supports the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic of the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblast (CAF) cells. Resulting cell patterns recapitulate key aspects of native tissue dynamics of the densely packed tumor tissues, in which CAF cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe an engineered synNotch-based signaling system in patterned cell patches that mimics morphogen gradient formation, where GFP-secreting sender cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architecturesinvitrowith biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.