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Automated hiPSC culture and sample preparation for 3D live cell microscopy
Open accessAbstract
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.
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Cells were regularly assessed for morphology, cell stemness marker expression and outsourced cytogenetic analyses throughout the three years of data acquisition.
“Cells were regularly assessed for morphology, cell stemness marker expression and outsourced cytogenetic analyses throughout the three years of data acquisition of the WTC-11 hiPSC Single-Cell Image Dataset v1 (ref. 34).”
From Integrated intracellular organization and its variations in human iPS cells (2023) · cites this paper as [34]
The Cell culture morphology and confluency section (page unavailable) says morphology was monitored every four days. The Stemness marker expression assessment and Cytogenetic stability analysis sections (pages unavailable) describe marker testing and karyotyping sent to Diagnostic Cytogenetics, Inc. Our copy does not establish that all three assessments were performed regularly throughout the dataset’s three years of acquisition. (LLM, conf=0.84)
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References 31 · Cited by 6
Metadata
- Authors
- Coston, M. E., Gregor, B. W., Arakaki, J., Borensztejn, A., Do, T. P., Fuqua, M. A., Haupt, A., Hendershott, M. C., Leung, W., Mueller, I. A., Nelson, A. M., Rafelski, S. M., Swain-Bowden, M. J., Tang, W. J., Thirstrup, D. J., Wiegraebe, W., Yan, C., Gunawardane, R. N., & Gaudreault, N.
- Affiliations
- Allen Institute for Cell Science (US)
- Corresponding author
- Gaudreault, Nathalie
- ORCID
- Venue
- bioRxiv (Cold Spring Harbor Laboratory)
- Publisher
- openRxiv
- Published
- 2020-12-19
- OpenAlex
- W3111191379
- Cited by
- 6
- References
- 31
- Citations by year
- 2024: 1 · 2023: 2 · 2022: 1 · 2021: 1 · 2020: 1
- Type
- posted content
- Language
- en
- Open access
- green
- doi.org
- Locations
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Keywords
- human induced pluripotent stem cells, automated cell culture, 3D live-cell imaging, high-content screening, quality control
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