Project Summary The hierarchical arrangement of cells within tissue plays an important role in determining function. As part of this hierarchical arrangement, different cell types are spatially arranged in contact with one another in a way that transmits important signaling cues that direct a multitude of different functional and dysfunctional cellular responses, such as altered gene expression, migration, metabolite sharing, and survival. A greater understanding of how cell arrangement impacts these behaviors would have important repercussions for a host of developmental processes that include stem cell differentiation, cancer metastasis, scar tissue formation, immunology, and angiogenesis. While the importance of spatially-regulated hierarchical cell arrangements is well established, methods for reproducing this complexity with high precision remain limited. Conventionally, model organisms have informed much of what is understood about these processes, but often do not allow constant direct observation and control. Rapidly evolving 3D printing methods have greatly enhanced our ability to place cells on substrates with libraries of different materials. However, these technologies do not allow one to precisely place individual cells in contact with each other in order to understand how different arrangements drive biological processes in highly heterogenous cell populations. Likewise, techniques that facilitate cell-cell contact placement do not readily enable 3D control with multiple different cell types. This proposal seeks to establish the feasibility of technology that would address this biomedical technological need. Specifically, it evaluates the use of oligonucleotide (short DNA sequences) to precisely control cell placement in an interchangeable and on-the- fly fashion. Aim 1 of this proposal seeks to establish new methods and design rules for dynamically and sequentially adding multiple different cells to a surface with high fidelity and spatial control. Aim 2 seeks to develop a new approach to building spatially controlled 3D cell assemblies using programmable DNA. Completion of this proposal will establish feasibility of this technology for future applications in biomedical studies.