TY - JOUR T1 - Automated hiPSC culture and sample preparation for 3D live cell microscopy JF - bioRxiv DO - 10.1101/2020.12.18.423371 SP - 2020.12.18.423371 AU - Mackenzie E. Coston AU - Benjamin W. Gregor AU - Joy Arakaki AU - Antoine Borensztejn AU - Thao P. Do AU - Margaret A. Fuqua AU - Amanda Haupt AU - Melissa C. Hendershott AU - Winnie Leung AU - Irina A. Mueller AU - Angelique M. Nelson AU - Susanne M. Rafelski AU - Madison J. Swain-Bowden AU - W. Joyce Tang AU - Derek J. Thirstrup AU - Winfried Wiegraebe AU - Calysta Yan AU - Ruwanthi N Gunawardane AU - Nathalie Gaudreault Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/12/19/2020.12.18.423371.abstract N2 - Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.Competing Interest StatementThe authors have declared no competing interest. ER -