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Double-Barrel Perfusion System for Modification of Luminal Contents of Intestinal Organoids

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3D Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2764))

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Abstract

Organoids are 3D cultures of self-organized adult or pluripotent stem cells with an epithelial membrane enclosing a defined fluid-filled lumen. These organoids have been demonstrated with a wide range of organotypic tissue types, but the enclosed nature of the structure restricts access to the lumen and apical surface of the cell membrane. To increase the potential applications of organoids, new technologies are required to provide access to the lumen of the organoid and apical surface of the epithelial cell membrane to enable new biomedical studies. This chapter details a method to access the lumen and apical surface of an organoid utilizing a double-barrel pulled glass capillary and pressure-based pump. The organoid perfusion system uses a three-axis micromanipulator to position the double-barrel capillary to pierce the organoid with the tip of the capillary. Each barrel of the double-barrel capillary is controlled independently with the pressure-based pump to allow injection and removal of material into and from the lumen. Additionally, the organoid is immobilized with a custom-designed PDMS organoid holder. The design of the components for the organoid perfusion system and details on their use are presented here and can be utilized as the basis to enable a wide range of organoid studies including but not limited to modifying luminal contents and apical cell membrane interactions during organoid cultures, recapitulation of physiological flow within the normally static organoid lumen, and effects of mechanical strain on organoid cell development.

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Acknowledgments

The authors would like to thank Professor Shuichi Takayama for supporting and developing these methods, Ge-Ah Kim for aiding with experiments and imaging of organoids, and Professor Jason Spence, Professor Vincent B. Young, Sha Huang, and Veda K. Yadagiri for supplying organoids for perfusion experiments and for invaluable discussions. This material is based upon work supported by the National Institute of Health (NIH) (Grant Number: U19 AI116482 and R01 HL136141).

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Authors and Affiliations

  1. Department of Mechanical & Aerospace Engineering, The University of Alabama in Huntsville, Huntsville, AL, USA

    Nicholas J. Ginga

  2. Department of Mechanical Engineering, Stanford University, Stanford, CA, USA

    Raleigh Slyman

Authors
  1. Nicholas J. Ginga
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  2. Raleigh Slyman
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Corresponding author

Correspondence to Nicholas J. Ginga .

Editor information

Editors and Affiliations

  1. Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

    Zuzana Sumbalova Koledova

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Ginga, N.J., Slyman, R. (2024). Double-Barrel Perfusion System for Modification of Luminal Contents of Intestinal Organoids. In: Sumbalova Koledova, Z. (eds) 3D Cell Culture. Methods in Molecular Biology, vol 2764. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3674-9_14

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  • DOI: https://doi.org/10.1007/978-1-0716-3674-9_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3673-2

  • Online ISBN: 978-1-0716-3674-9

  • eBook Packages: Springer Protocols

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