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Automated hiPSC culture and sample preparation for 3D live cell microscopy

Mackenzie E. Coston, Benjamin W. Gregor, Joy Arakaki, Antoine Borensztejn, Thao P. Do, Margaret A. Fuqua, Amanda Haupt, Melissa C. Hendershott, Winnie Leung, Irina A. Mueller, Angelique M. Nelson, Susanne M. Rafelski, Madison J. Swain-Bowden, W. Joyce Tang, Derek J. Thirstrup, Winfried Wiegraebe, Calysta Yan, Ruwanthi N Gunawardane, Nathalie Gaudreault
doi: https://doi.org/10.1101/2020.12.18.423371
Mackenzie E. Coston
1 Allen Institute for Cell Science;
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Benjamin W. Gregor
1 Allen Institute for Cell Science;
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Joy Arakaki
1 Allen Institute for Cell Science;
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Antoine Borensztejn
1 Allen Institute for Cell Science;
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Thao P. Do
1 Allen Institute for Cell Science;
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Margaret A. Fuqua
1 Allen Institute for Cell Science;
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Amanda Haupt
1 Allen Institute for Cell Science;
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Melissa C. Hendershott
1 Allen Institute for Cell Science;
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Winnie Leung
1 Allen Institute for Cell Science;
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Irina A. Mueller
1 Allen Institute for Cell Science;
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Angelique M. Nelson
1 Allen Institute for Cell Science;
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Susanne M. Rafelski
2 Allen Institute for Cel Science
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Madison J. Swain-Bowden
1 Allen Institute for Cell Science;
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W. Joyce Tang
1 Allen Institute for Cell Science;
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Derek J. Thirstrup
1 Allen Institute for Cell Science;
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Winfried Wiegraebe
1 Allen Institute for Cell Science;
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Calysta Yan
1 Allen Institute for Cell Science;
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Ruwanthi N Gunawardane
1 Allen Institute for Cell Science;
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Nathalie Gaudreault
1 Allen Institute for Cell Science;
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  • For correspondence: nathalieg@alleninstitute.org
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Abstract

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 Statement

The authors have declared no competing interest.

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Posted December 19, 2020.
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Automated hiPSC culture and sample preparation for 3D live cell microscopy
Mackenzie E. Coston, Benjamin W. Gregor, Joy Arakaki, Antoine Borensztejn, Thao P. Do, Margaret A. Fuqua, Amanda Haupt, Melissa C. Hendershott, Winnie Leung, Irina A. Mueller, Angelique M. Nelson, Susanne M. Rafelski, Madison J. Swain-Bowden, W. Joyce Tang, Derek J. Thirstrup, Winfried Wiegraebe, Calysta Yan, Ruwanthi N Gunawardane, Nathalie Gaudreault
bioRxiv 2020.12.18.423371; doi: https://doi.org/10.1101/2020.12.18.423371
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Automated hiPSC culture and sample preparation for 3D live cell microscopy
Mackenzie E. Coston, Benjamin W. Gregor, Joy Arakaki, Antoine Borensztejn, Thao P. Do, Margaret A. Fuqua, Amanda Haupt, Melissa C. Hendershott, Winnie Leung, Irina A. Mueller, Angelique M. Nelson, Susanne M. Rafelski, Madison J. Swain-Bowden, W. Joyce Tang, Derek J. Thirstrup, Winfried Wiegraebe, Calysta Yan, Ruwanthi N Gunawardane, Nathalie Gaudreault
bioRxiv 2020.12.18.423371; doi: https://doi.org/10.1101/2020.12.18.423371

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