Name: an intestinal gut organ culture system for analyzing host-microbiota interactions
Uploaded: 2021-06-30T15:00:00
Description: Watch this Scientific Journal Video about An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions at JoVE.com

We thank past and present members of the Yissachar lab for their valuable contributions in optimizing the gut organ culture system protocol. We thank Yael Laure for critical editing of the manuscript. This work was supported by the Israel Science Foundation (grant No. 3114831), the Israel Science Foundation - Broad Institute Joint Program (grant No. 8165162), and the Gassner Fund for Medical Research, Israel.

This protocol follows the animal care guidelines approved by the ethics committee for animal welfare.
1. Experiment preparation
<ol>
	<li>Fabrication of the gut organ culture device (3 days)
	<ol>
		<li>Using a 3D printer, print the reusable plastic molds for the organ culture device (the device has 6 wells, with 24 small and large holes, and for the device cover lid) (3D files attached). 
		NOTE: These plastic molds may be used for fabrication of numerous devices.</li>
		<li>Insert the...

<ol>
	<li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews Immunology. 14 (10), 667-685 (2014).</li><li>Pearce, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+in+vitro+and+ex+vivo+Models+to+Study+Host-Microbiome+Interactions+and+Acute+Stressors.">Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors.</a> Frontiers in Physiology. 9 (1584), (2018).</li><li>Hooper, L. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+commensal+host-microbial+relationships+in+the+intestine.">Molecular analysis of commensal host-microbial relationships in the intestine.</a> Science. 291 (5505), 881-884 (2001).</li><li>Haller, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-pathogenic+bacteria+elicit+a+differential+cytokine+response+by+intestinal+epithelial+cell/leucocyte+co-cultures.">Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.</a> Gut. 47 (1), 79-87 (2000).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. 61 (7), 1007-1015 (2012).</li><li>Gazzaniga, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+Colon+Chip+Technology+to+Identify+Commensal+Bacteria+That+Promote+Host+Tolerance+to+Infection.">Harnessing Colon Chip Technology to Identify Commensal Bacteria That Promote Host Tolerance to Infection.</a> Frontiers in Cellular and Infection Microbiology. 11, 638014 (2021).</li><li>Yissachar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Intestinal+Organ+Culture+System+Uncovers+a+Role+for+the+Nervous+System+in+Microbe-Immune+Crosstalk.">An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.</a> Cell. 168 (6), 1135-1148 (2017).</li><li>Duscha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propionic+Acid+Shapes+the+Multiple+Sclerosis+Disease+Course+by+an+Immunomodulatory+Mechanism.">Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.</a> Cell. 180 (6), 1067-1080 (2020).</li><li>Grosheva, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screen+Identifies+Host+and+Microbiota+Regulators+of+Intestinal+Barrier+Function.">High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.</a> Gastroenterology. 159 (5), 1807-1823 (2020).</li><li>Blaize, J. F., Corbo, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+Dilutions+and+Plating:+Microbial+Enumeration.">Serial Dilutions and Plating: Microbial Enumeration.</a> Journal of Visualized Experiments. , (2021).</li><li>Ivanov, I. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+intestinal+Th17+cells+by+segmented+filamentous+bacteria.">Induction of intestinal Th17 cells by segmented filamentous bacteria.</a> Cell. 139 (3), 485-498 (2009).</li><li>Schnupf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+host+interaction+of+mouse+segmented+filamentous+bacteria+in+vitro.">Growth and host interaction of mouse segmented filamentous bacteria in vitro.</a> Nature. 520 (7545), 99-103 (2015).</li><li>Chung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+immune+maturation+depends+on+colonization+with+a+host-specific+microbiota.">Gut immune maturation depends on colonization with a host-specific microbiota.</a> Cell. 149 (7), 1578-1593 (2012).</li><li>Atarashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+Cell+Induction+by+Adhesion+of+Microbes+to+Intestinal+Epithelial+Cells.">Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.</a> Cell. 163 (2), 367-380 (2015).</li></ol>

This article describe an optimized protocol for ex vivo gut organ cultures that Yissachar et al. have recently developed (published9 and unpublished data). The gut organ culture system supports multiplexed culture of intact intestinal fragments while maintaining luminal flow. It provides full control over the intra- and extra-luminal environment (including stimulation dose, exposure time and flow rate) and preserves the na&#239;ve intestinal tissue structure and its cellular complexity<su...

The intestine is a highly complex organ containing a wide range of cell types (epithelial cells, immune system cells, neurons, and more) organized in a particular structure that allows cells to interact and communicate with one another and with the luminal content (microbiota, food, etc.)1. Currently, the research toolbox available for analyzing host-microbiota interactions includes in vitro cell cultures and in vivo animal models2. In vivo animal models provide a physiological tissue construction3 but with poor experimental control and limited ability to manipulate the experiment conditions. In vitro culture systems, on the other hand, use primary cells or cell lines that can be supplemented with microbes4, offering tight control over the experiment parameters but lack the cellular complexity and the tissue architecture. Modern in vitro assays allow the advanced use of healthy and pathological human tissue samples, like epithelial organoids derived from mouse or human sources5,6, and samples that mimic the mucosal microenvironment7. Another example is the &#39;gut on a chip&#39; assay, which includes the human colonic epithelial cell line (Caco2), extracellular matrix and microfluidic channels to mimic the physiological condition of the gut invariant8. However, as advanced and innovative as in vitro samples may be, they do not maintain a normal tissue architecture or na&#239;ve cellular composition.
To address that, Yissachar et al. recently developed an ex vivo organ culture system9 (Figure 1)&#160;that maintains intact gut fragments ex vivo, benefiting from the advantages of both in vivo and in vitro models. This ex vivo gut organ culture system is based on a custom-made culture device that supports a multiplexed culture of six colon tissues, allowing examining experimental inputs under comparable conditions while controlling the system&#39;s inputs and outputs. Recent works have demonstrated that this system is valuable for analyzing intestinal responses to individual gut bacteria9, whole human microbiota samples10 and microbial metabolites11. This system allows, for the first time, the study of these early host-microbiota interactions with a high level of control over the host, microbial and environmental components. Furthermore, it allows monitoring and manipulating the system throughout the experiment, in real-time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62779/62779fig01.jpg" /> 
Figure 1: Schematics of the gut culture device. A whole intestinal tissue fragment is attached to the output and input ports of the chamber (top), with pumps regulating the medium flow inside the lumen and in the external medium chamber. The entire device (bottom) contains 6 such chambers. This figure has been modified from Yissachar et al. 2017. <a href="https://www.jove.com/files/ftp_upload/62779/62779fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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			<td>Polydimethylsiloxane, PDMS</td>
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			<td>Tubing</td>
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			<td>Zortrax M200 Materials: z-ultrat</td>
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			<td>Medium</td>
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			<td>B27 Supplement (50x), Serum Free</td>
			<td>Thermo Fisher Scientific</td>
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			<td>HEPES Buffer (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630056</td>
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			<td>Iscove&#39;s Mod Dulbecco&#39;s Medium With Phenol Red (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440061</td>
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			<td>Knock-Out Serum</td>
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an intestinal gut organ culture system for analyzing host-microbiota interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

The gut organ culture system maintains tissue viability ex vivo. The evaluation of the tissue viability was done throughout the culture period. Colon tissue fragments were incubated in the gut organ culture system and fixed following 2/12/24 h culture. The intestinal epithelial cell (IEC) layer integrity was validated by immunofluorescence staining using E-cadherin and cytokeratin-18 antibodies. Likewise, mucus-filled goblet cells in the colonic epithelium and mucus secretion within the lumen were detected as we...

Watch this Scientific Journal Video about An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions at JoVE.com

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

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