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>
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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. 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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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Device</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>18 Gauge Blunt Needle</td>
			<td>Mcmaster</td>
			<td>75165a754</td>
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			<td>22 Gauge Blunt Needle</td>
			<td>Mcmaster</td>
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			<td>All Purpose Adhesive Selant 100% Silicone</td>
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			<td>Cubic Vacuum Desiccator VDC-21+ 2 Shelves</td>
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			<td>MPC 301E Vacuum PUMP</td>
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			<td>VI-412711</td>
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			<td>Plastic Quick Turn Tube Coupling Plugs</td>
			<td>Mcmaster</td>
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			<td>plastic Quick Turn Tube Coupling Sockets</td>
			<td>Mcmaster</td>
			<td>52525k211</td>
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			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Dow</td>
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			<td>Polydimethylsiloxane, PDMS</td>
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			<td>Tubing</td>
			<td>Mcmaster</td>
			<td>6516t11</td>
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			<td>Zortrax M200</td>
			<td>Zortrax</td>
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			<td>Zortrax Z-SUITE, Autodesk Fusion 360</td>
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			<td>Zortrax M200 Materials: z-ultrat</td>
			<td>Zortrax</td>
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			<td>Medium</td>
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			<td>B27 Supplement (50x), Serum Free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
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			<td>HEPES Buffer (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Mod Dulbecco&#39;s Medium With Phenol Red (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440061</td>
			<td></td>
		</tr>
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			<td>Knock-Out Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
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			<td>N2 Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1370701</td>
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			<td>Non Essential Amino Acid (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
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			<td>Surgical Tools</td>
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			<td>Large Scissors</td>
			<td>Aseltech</td>
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			<td>Sharp Forceps</td>
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			<td>Silk Braided Surgical Thread</td>
			<td>SMI</td>
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			<td>F.S.T</td>
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			<td>Thin Forceps</td>
			<td>F.S.T</td>
			<td>11051-10</td>
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			<td>Organ System</td>
			<td></td>
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			<td>0.1 &#181;m Filter</td>
			<td>Life Gene</td>
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			<td>0.22 &#181;m Filter</td>
			<td>Life Gene</td>
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			<td>5 mL Luer Lock Syringe</td>
			<td>B-D</td>
			<td>309649</td>
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			<td>Greenough Stereo Microscope</td>
			<td>ZEISS</td>
			<td>Stemi 305</td>
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			<td>Recirculating Precision Air Heater &#34;CUBE&#34;</td>
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			<td>CUBE-2-LIS</td>
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			<td>new era pump systems inc</td>
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</table>

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 ...

The gut organ culture system combines the advantages of in vitro and in vivo assays and thus serves as an intermediate experimental step between simple in vitro assays to complex in vivo experiments. The main advantage of this technique is the combination of tight experimental control with the preservation of intestinal physiology. This method provides insight for analyzing intestinal responses to specific stimulus with high level of control over the host, microbial, and the environmental components.Demonstrating the procedure will be Alon Shemesh, a Masters student from my laboratory, and Dr.Sivan Amidror, our lab manager. To begin, insert the blunt-end needles to the appropriate position within the device mold and cast approximately 20 grams of polydimethylsiloxane or PDMS mix, for one set of the device and lid. Place the molds in a vacuum chamber for 30 minutes to remove air bubbles from the PDMS mix, then incubate the molds at 55 degrees Celsius overnight to complete PDMS polymerization.When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds. Remove PDMS residues from the well outline using a surgical blade. Attach the PDMS device and the device cover onto a cover glass of 75 by 50 millimeter microslides using non-toxic silicon adhesive and leave the parts to set overnight.Apply the glued to the smooth side of the device. Insert 12, 22 gauge needles for the lumen and 12, 18 gauge needles for the well. Fix all the needles in place using silicone and let it set overnight.Purge the input syringes and make sure that the well medium flows out from all tubes into a waste glass, then purge the input syringes and makes sure that the stimulations flow out of all the tubes into a waste glass, taking care to not contaminate the different stimulations. After sacrificing the mouse, use sharp scissors and forceps to dissect it and take out the digestive tract from the stomach to the anus, by cutting all the fat and connective tissues. Cut the colon and place it on a new plate.Minimize contact while holding the tissue gently and only at the edges. Perform the colon flush under a dissection microscope. Gently flush the colon content with sterile IMDM with the prepared 10 milliliter syringe as described in the text.After removing the feces from the intestinal tissue, place the colon in a new six well plate filled with 0.5 milliliters of sterile IMDM. Next, take the colon tissue and carefully connect it to the 22 gauge needle, making a tight tie with the two threads. Maintain the correct orientation of the colon to the lumen flow, such that the proximal and distal is equal to input and output, respectively.Repeat colon flushing and needle tying for all the tissues. Connect the input and output tubes to the device, then begin the experiment by starting the pumps at the desired rates. Mucus-filled goblet cells in the colonic epithelium and mucus secretion within the lumen were detected as well as proliferating IEC in the colonic crypts, as indicated by KI67 staining.These results showed that the gut culture system maintains intestinal function and structure ex-vivo. After two hours of segmented filamentous bacteria, or SFB, introduction, typical SFB filaments were detected in close association with small intestine villi using fluorescence in situ hybridization. Additionally, a transmission electron microscopy presented SFB within a few microns of the small intestine epithelium brush border.Gene expression profiles of whole tissue samples were produced in triplicate two hours after infusion with SFB. Control cultures were infused with fecal suspensions of germ-free or Bacteroides fragilis monocolonized mice. These changes persuaded by SFB were mainly of small amplitude compared to the germ-free control.The orientation of the colon is highly important. Take extra care when tying the colon on the needle. Proper tie will prevent the contamination of the well with the lumen content.A wide range of readout techniques can follow this protocol, such as next generation sequencing, imaging, cell sorting, and many more. This readouts provide novel insight into host microbiome interactions in health and disease.

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Immunology and Infection

4.0K visualizações.  Bar-Ilan University. O sistema de cultura de órgãos intestinais combina as vantagens dos ensaios in vitro e in vivo e, portanto, serve como um passo experimental intermediário entre ensaios in vitro simples e complexos. A principal vantagem dessa técnica é a combinação de controle experimental apertado com a preservação da fisiologia intestinal. Este método fornece insights para a análise de respostas intestinais a estímulos específicos com alto nível de controle sobre o hospedeiro, microbiano e os ...