Controlling the spatial organization of cells in 3D biological hydrogels for the study of cell-cell mechanical interactions

Methods to accurately pattern cells on 2D substrates are well-established, enabling a variety of applications in cell biology, tissue engineering and drug screening. However, cell micropatterning in biomimetic 3D environments has been more challenging, especially at the micro-scale resolution, and currently relies on sophisticated tools and protocols. We develop here a simple and effective method (> 90% efficiency) to micropattern large arrays of single cells or cell spheroids of defined sizes in biological hydrogels, with precise control over cell-cell distances and neighboring cells position. We utilize a silicone strip to mechanically support the gel and facilitate transfer of cells from a glass substrate to a 3D gel by peeling-off the gel with the silicone carrier. We used these large gel patterns to study the long-range, matrix-mediated mechanical interactions between well-organized array of cells, a process relevant to tissue morphogenesis, tumor development and wound healing. At 200 µm cell-cell spacing, we identified a collective formation of intense cell-generated anisotropic tension lines emerging in the gel between near neighbors, along which cells preferentially migrate. Beyond revealing fundamental cell morphogenetic processes, the presented gel micropatterning method can be used to reconstruct any desired tissue geometry with micrometer resolution in 3D hydrogel environments, leveraging the engineering of tissues in complex architectures.