PROJECT SUMMARY Key to multicellularity is the coordinated interaction of the various cells that make up the body. Indeed, patterning of embryos, establishment of cell type diversity, and formation of tissues and organs all rely on cell-to-cell communication. Thus, arguably one of the most important principles of biology involves “one group of cells changing the behavior of an adjacent set of cells, causing them to change their shape, mitotic rate, or fate”. Conventional methods of reproducing biological patterns and cell-fate in vitro suffer from multiple limitations. Previous work on understanding pattern formation has relied on delivering global stimuli and studying reaction- diffusion mediated patterning of cell fates in the cell culture. Another method has been to generate morphogen gradients using signaling molecule patterned surface or optogenetics. However, all current methods produce static patterns and give neither precise spatial nor temporal control over the cell fate. My research group aims to overcome this critical challenge, via a unique and novel cyber-bio system, in which microrobots direct the biological system, in a closed loop approach, to enable position-specific functionality and reduce noise – to direct cellular fate leading to the formation of cellular structures. Inspired by “human-in-the loop” approaches for engineering systems that must interact with complex, living individuals, we propose a “µrobot-in-the-loop” approach in which physical signaling among cells is substituted with microrobot-controlled inputs to afford excellent spatiotemporal precision and feedback control in directing cell behavior. Our efforts in the next five years would focus on designing and fabricating microrobots along with developing control algorithms for automated actuation of the microrobots. We will use these microrobots to deliver morphogens at precise positions in a cellular system which would alter cell fate at those positions only. We would also use this technology for controlling the formation of multilayer cellular structures. We would extend this to three dimensional tissues by interfacing microrobots with organoids. The proposed work is important because it would demonstrate how individual cells in a tissue volume can be spatially and temporally targeted for manipulation. This methodology applies more dynamic control over differentiation factors, which allows for increased understanding of complicated cell fate and differentiation events during cancer, development, or fibrosis as just a few of many applications.