PROJECT SUMMARY In the last 20 years, both academia and industry have significantly shifted from target-based drug discovery to phenotypic screening. An unmet need in the biomedical research enterprise has long been human disease models with reliable translational content that can be used to screen small molecule or RNAi/gRNA/ORF libraries for desirable phenotype changes. Recent advances in culturing organoids or 3D tissue derived from patient or progenitor cells provide opportunities for meeting this challenge. It is widely accepted that drug discovery using such in vitro models represents an accelerated route to pre-clinical testing of drug candidates because organoids and 3D structures more closely resemble the complex physiopathology of diseased tissue than cultured cell lines or biochemical assays. An ongoing challenge in the field is the upgrading of instrumentation, technology, and methods to visualize and measure components and features (phenotypes) in responses to molecular treatment within these 3D models. The Vanderbilt Institute of Chemical Biology and Vanderbilt High-throughput Screening (VHTS) facility is a well- established academic drug discovery center that uses innovative technologies to advance scientific and clinical therapeutic discoveries. Currently, the VHTS facility has an automated fluorescence-based microscope for high- content imaging (HCI) and phenotypic screening that is integrated with a robot arm and incubator, which allow for automated live and fixed cell imaging in micro-titer plates. Studies that have utilized this system include iPSC- derived cell screens, functional genomics screens (siRNA, ORF), mechanisms of viral replication, drug toxicity studies, drug screens for novel therapeutic development, drug mechanisms of action and resistance, and drug combination studies. However, due to the lack of confocal imaging capability, this system is severely inadequate for imaging and measuring features within organoids and 3D structures. In addition, the current vendor no longer supports service of the robot arm and incubator. Therefore, it is critical to acquire a new automated HCI system, complete with robotic equipment, in order to enable state-of-the-art high-throughput phenotypic screening approaches to disease biology and drug discovery using organoids and 3D structures. The proposed HCI drug discovery system will center on a Molecular Devices (MD) ImageXpress Micro Confocal HCI system, with integration of a Precise Automation PreciseFlex PF400 robot arm and Liconic automated incubator. This combination of instruments will allow for high-throughput phenotypic screening at every desired level and capacity. We note that MD has committed to designing the most robust automated microscope instrument with the intention of implementing optimization for both speed and accuracy specifically for HCI of 3D structures and organoids. The proposed system would add significant value to the already established Vanderbilt clientel...