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Organoids pp 215–228Cite as

Intestinal Crypt Organoid: Isolation of Intestinal Stem Cells, In Vitro Culture, and Optical Observation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1576))

Abstract

The isolation and culture of intestinal stem cells (ISCs) was first demonstrated in the very recent decade with the identification of ISC marker Lgr5. The growth of ISCs into crypt organoids provides an in vitro model for studying the mucosal physiology, intestinal cancer tumorigenesis, and intestinal regeneration. Here, we describe two different isolation protocols and demonstrate a fixation method that aids in the confocal observation of the organoids.

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Author information

Authors and Affiliations

  1. Department of Surgery, Far Eastern Memorial Hospital, Pan-Chiao, New Taipei, Taiwan

    Yun Chen & Ya-Hui Tsai

  2. Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li, Taoyuan, Taiwan

    Yun Chen & Ya-Hui Tsai

  3. Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan

    Chuan Li

  4. Department of Surgery, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan

    Sheng-Hong Tseng

Authors
  1. Yun Chen
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  2. Chuan Li
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  3. Ya-Hui Tsai
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  4. Sheng-Hong Tseng
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Corresponding authors

Correspondence to Ya-Hui Tsai or Sheng-Hong Tseng .

Editor information

Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, ON, Canada

    Kursad Turksen

1 Electronic Supplementary Material

3-D reconstruction for the organoids. The organoids were stained with DAPI and the fluorescence signals were imaged by confocal microscopy under the 40× oil objective. The Z-stacked images were processed and reconstructed by Zen software (Carl Zeiss). This video covered part of the organoid with seven visible crypt-like domains (AVI 2061 kb)

3-D reconstruction for the organoids. The organoids were stained with DAPI and the fluorescence signals were imaged by confocal microscopy under the 63× oil objective. The Z-stacked images were processed and reconstructed by Zen software (Carl Zeiss). This video covered only one crypt-like domain of an organoid (AVI 2440 kb)

Video 3

The growth of ISC into crypt organoids. The images were captured every 8 h on the first day and every 24 h afterward for 20 days. The images were captured and recorded by AZTEC CCM-1.4 II/M. The scale bar in the video represents 100 μm (AVI 31107 kb)

Video 4

The growth of organoids after passage. The images were captured every 24 h after passage for 9 days. The images were captured and recorded by AZTEC CCM-1.4 II/M. The scale bar in the video represents 100 μm (AVI 12728 kb)

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Chen, Y., Li, C., Tsai, YH., Tseng, SH. (2017). Intestinal Crypt Organoid: Isolation of Intestinal Stem Cells, In Vitro Culture, and Optical Observation. In: Turksen, K. (eds) Organoids. Methods in Molecular Biology, vol 1576. Humana, New York, NY. https://doi.org/10.1007/7651_2017_21

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  • DOI: https://doi.org/10.1007/7651_2017_21

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-7616-4

  • Online ISBN: 978-1-4939-7617-1

  • eBook Packages: Springer Protocols

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