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

Protocol
Part of the Methods in Molecular Biology book series

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.

Keywords:

Intestinal stem cell (ISC) Crypt organoid Isolation Confocal observation Three-dimensional imaging 

Supplementary material

7651_2017_21_MOESM1_ESM.avi (2 mb)
Video 13-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)
7651_2017_21_MOESM2_ESM.avi (2.4 mb)
Video 23-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)
7651_2017_21_MOESM3_ESM.avi (30.4 mb)
Video 3The 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)
7651_2017_21_MOESM4_ESM.avi (12.4 mb)
Video 4The 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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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of SurgeryFar Eastern Memorial HospitalNew TaipeiTaiwan
  2. 2.Department of Chemical Engineering and Materials ScienceYuan Ze UniversityTaoyuanTaiwan
  3. 3.Department of Biomedical EngineeringNational Yang-Ming UniversityTaipeiTaiwan
  4. 4.Department of SurgeryNational Taiwan University Hospital, National Taiwan University College of MedicineTaipeiTaiwan

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