Name: an intestinal gut organ culture system for analyzing host-microbiota interactions
Uploaded: 2021-06-30T15:00:00.000+00:00
Duration: 5 min 27 s
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 blunt-end needles (22 G &#38; 18 G) to the appropriate position within the device mold and cast approximately 20 g of polydimethylsiloxane (PDMS) mix (1:10 weight ratio, base to catalyst) for one set of the device and lid.</li>
		<li>Place the molds in a vacuum chamber for 30 min, to remove air bubbles from the PDMS mix.</li>
		<li>Incubate the molds at 55 &#176;C overnight, to complete PDMS polymerization.</li>
		<li>When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds.</li>
		<li>Remove PDMS residues from the well outline using a surgical blade. Attach the PDMS device and the device cover onto a cover glass (75 mm x 50 mm micro slides) using non-toxic silicon adhesive and leave the parts to set overnight (apply the glue to the smooth side of the device).</li>
		<li>Insert twelve 22 G needles for the lumen and twelve 18 G needles for the well. Fix all the needles in place using silicone and let it set overnight. Insert two 18 G needles into the cover lid for proper air flow in and out of the gut organ culture device.</li>
		<li>Check all the needles for leaks using a water-filled syringe. Check that there is no leaking from the wells by filling the wells with water.</li>
		<li>Place two surgical knots on each 22 G needle that will be connected to the colon. Place the device and the lid in an autoclave paper bag and sterilize in an autoclave.</li>
	</ol>
	</li>
	<li>Culture medium (0.5 h)
	<ol>
		<li>In a biological hood, mix the following (in 50 mL tube): 37 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), 10 mL of KSR serum replacement, 1 mL of B27&#160;supplement, 0.5 mL of N2 supplement, 0.5 mL of 1 M HEPES buffer, and 0.5 mL of non-essential amino acids.</li>
		<li>Filter, and store the complete medium at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Tubing and surgical tool preparation
	<ol>
		<li>Cut the appropriate length of tubes for the input lumen, input well, output lumen, and output well (12 short tubes and 12 long tubes). Connect an appropriate adaptor to each side of the tube.</li>
		<li>Prepare the surgical tools: straight scissors, 4x thin forceps, and 2x sharp forceps.</li>
		<li>Place the tubes and the surgical tools in an autoclave paper bag and sterilize them using an autoclave.</li>
	</ol>
	</li>
	<li>Prepare the luminal input (desired stimulation - bacteria, stool, drugs, etc.).
	<ol>
		<li>Before the experiment, determine the bacterial load of the bacterial culture, by serial dilutions12, and culture under aerobic and/or anaerobic conditions.</li>
		<li>After calculating the bacterial load, dilute the bacterial cultures in sterile tissue culture medium to obtain the required bacterial concentration. For fecal samples, filter using a 100 &#181;m strainer. 
		&#8203;NOTE: For nonbacterial stimulation (drugs, metabolites, etc.), dilute the substance to the required concentration using the gut culture medium.</li>
	</ol>
	</li>
</ol>
2. Experiment setup preparation
<ol>
	<li>Inside the organ culture incubator, turn on the heater unit, and set it to 37 &#176;C.</li>
	<li>Set up the pumps as well as the input and output syringes.</li>
	<li>Tissue culture medium input
	<ol>
		<li>In a biological or laminar flow hood, fill the input well syringes with complete culture medium. The final volume depends on the experiment&#39;s duration and the flow rate; usually 1 mL/h plus additional medium for purge.</li>
		<li>Connect the tubes to the syringes using the Luer-lock adaptor. Place the filled syringes in the syringe pumps.</li>
		<li>Purge the input syringes. Make sure that the well medium flows out from all tubes into a waste glass.</li>
	</ol>
	</li>
	<li>Luminal input
	<ol>
		<li>Fill the luminal input syringes with stimulation treatment (bacteria, drugs, etc.). The volume depends on the duration of the experiment and the flow rate (usually 30 &#181;L/h plus additional medium for purge).</li>
		<li>Connect the tubes to the syringes using the Luer-lock adaptor. Place the filled syringes in the syringe pumps.</li>
		<li>Purge input syringes. Make sure that the stimulation flow out of all the tubes into a waste glass. Be careful not to contaminate the different stimulations.</li>
	</ol>
	</li>
	<li>Outputs
	<ol>
		<li>Place the empty syringes in the output syringe pumps (set pump mode to &#39;withdrawal&#39;).</li>
	</ol>
	</li>
	<li>Device setup
	<ol>
		<li>Set the regulator that controls the gas mixture (95% O2 + 5% CO2) flow rate for a gentle, minimal flow.</li>
		<li>Examine the device in a laminar hood.</li>
		<li>Flush the needles of the device with sterile IMDM to wash the needles.</li>
		<li>Add 500 &#181;L of sterile culture medium into each well of the device.</li>
	</ol>
	</li>
	<li>Preparation for tissue dissection
	<ol>
		<li>Put the sterile surgical tools inside the laminar hood.</li>
		<li>Fill a 10 mL syringe with sterile IMDM and connect a sterile (autoclaved) 22 G blunt-end needle for flushing of the colon.</li>
	</ol>
	</li>
</ol>
3. Organ cultures
<ol>
	<li>Mice sacrifice and tissue dissection
	<ol>
		<li>In a laminar hood, sacrifice 12-14 day old mice by decapitation. Spray the mice with 70% ethanol and place the mice on a plastic plate.</li>
		<li>Using sharp scissors and forceps, dissect the mouse 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. 
		&#8203;NOTE: Minimize the contact with the colon tissue. Do not touch the middle part of the colon tissue. Hold the tissue gently and only at the edges of the tissue.</li>
	</ol>
	</li>
	<li>Colon flush and wash
	<ol>
		<li>Under a dissection microscope, gently flush the colon content with sterile IMDM (into the proximal side) with the prepared 10 mL syringe (step 2.7.2). After removing the feces from the intestinal tissue, place the colon in a new 6 well plate filled with 0.5 mL of sterile IMDM.</li>
	</ol>
	</li>
	<li>Connecting the colon to the device
	<ol>
		<li>Take the tissue and carefully connect it onto the 22 G needle and make a tight tie with the two threads. At this point, it is imperative to maintain the correct orientation of the colon to the lumen flow (proximal = input, distal = output). Repeat steps 3.2-3.3 for all 6 tissues.</li>
		<li>Verify that the input needle Luer locks are empty from the medium. If not, empty them. Add stimulations to the input needle Luer lock (to avoid entry of air bubbles into the lumen). Repeat this step for each colon with the appropriate stimulation.</li>
		<li>Check that all the tissues are connected and place the cover lid on top of the device.</li>
	</ol>
	</li>
	<li>Connecting the organ culture device to the pumps
	<ol>
		<li>Place the device into the pre-heated temperature-controlled chamber (37 &#176;C).</li>
	</ol>
	</li>
	<li>Connect gas flow
	<ol>
		<li>Connect the gas adaptor to the cover lid using the appropriate input needle.</li>
		<li>Connect the input and output tubes to the device. 
		&#8203;NOTE: Connect the proximal colon side to the input tubs.</li>
	</ol>
	</li>
	<li>Purge the lumen with the stimulation inputs.
	<ol>
		<li>Gently flow luminal stimulation through the gut and verify medium flow in the output tubes.</li>
		<li>Wash external medium.</li>
		<li>Wash the external medium 3 times (set the pumps at a rate of 600 &#181;L/min for both input and output well). Each wash takes 1 min (starting by emptying the well).</li>
	</ol>
	</li>
	<li>Start the experiment.
	<ol>
		<li>Start the pumps at the following rates: 
		Flow rate: lumen: input- 30 &#181;L/h, output- 35 &#181;L/h 
		External medium: input- 1000 &#181;L/h, output- 950 &#181;L/h 
		&#8203;NOTE: Experiment time can vary between 30 min to 24 h.</li>
	</ol>
	</li>
	<li>End of experiment (up to 24 h for colon organ cultures)
	<ol>
		<li>Disconnect all the tubes from the device.</li>
		<li>Disconnect the tissues from the needles and continue to the desired readouts.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Device&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>18 Gauge Blunt Needle</td><td>Mcmaster</td><td>75165a754</td><td></td></tr><tr><td>22 Gauge Blunt Needle</td><td>Mcmaster</td><td>75165a758</td><td></td></tr><tr><td>All Purpose Adhesive Selant 100% Silicone</td><td>DAP</td><td>688</td><td></td></tr><tr><td>Cubic Vacuum Desiccator VDC-21+ 2 Shelves</td><td></td><td>AAAD4021</td><td></td></tr><tr><td>Glass Slide 1 mm Thick</td><td>Corning</td><td>2947-75X50</td><td></td></tr><tr><td>Mini Incubator im-10</td><td></td><td>AAH24315K</td><td></td></tr><tr><td>MPC 301E Vacuum PUMP</td><td></td><td>VI-412711</td><td></td></tr><tr><td>Plastic Quick Turn Tube Coupling Plugs</td><td>Mcmaster</td><td>51525k121</td><td></td></tr><tr><td>plastic Quick Turn Tube Coupling Sockets</td><td>Mcmaster</td><td>52525k211</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer</td><td>Dow</td><td></td><td>Polydimethylsiloxane, PDMS</td></tr><tr><td>Tubing</td><td>Mcmaster</td><td>6516t11</td><td></td></tr><tr><td>Zortrax M200</td><td>Zortrax</td><td></td><td>Zortrax Z-SUITE, Autodesk Fusion 360</td></tr><tr><td>Zortrax M200 Materials: z-ultrat</td><td>Zortrax</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B27 Supplement (50x), Serum Free</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>HEPES Buffer (1M)</td><td>Thermo Fisher Scientific</td><td>15630056</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium with Phenol Red (1x)</td><td>Thermo Fisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Knock-Out Serum</td><td>Thermo Fisher Scientific</td><td>10828028</td><td></td></tr><tr><td>N2 Supplement (100x)</td><td>Thermo Fisher Scientific</td><td>A1370701</td><td></td></tr><tr><td>Non Essential Amino Acid (100x)</td><td>Thermo Fisher Scientific</td><td>11140035</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Large Scissors</td><td>Aseltech</td><td>11-00-10</td><td></td></tr><tr><td>Sharp Forceps</td><td>F.S.T</td><td>11297-10</td><td></td></tr><tr><td>Silk Braided Surgical Thread</td><td>SMI</td><td>8010G</td><td></td></tr><tr><td>Straight Scissors</td><td>F.S.T</td><td>14091-09</td><td></td></tr><tr><td>Thin Forceps</td><td>F.S.T</td><td>11051-10</td><td></td></tr><tr><td>&lt;strong&gt;Organ System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.1 &amp;micro;m Filter</td><td>Life Gene</td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m Filter</td><td>Life Gene</td><td></td><td></td></tr><tr><td>5 mL Luer Lock Syringe</td><td>B-D</td><td>309649</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>ZEISS</td><td>Stemi 305</td><td></td></tr><tr><td>Recirculating Precision Air Heater &quot;CUBE&quot;</td><td></td><td>CUBE-2-LIS</td><td></td></tr><tr><td>Syringe Pump</td><td>new era pump systems inc</td><td>nep-ne-1600-em</td><td></td></tr></tbody></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 ...

an-intestinal-gut-organ-culture-system-for-analyzing-host-microbiota

Research

JoVE Journal

Immunology and Infection

4.1K Views.  Bar-Ilan University. This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments. 

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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Bioengineering

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biology

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Enhanced Intestine-on-Chip Platform for Immunological Insight

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...

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

Summary

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Chapters in this video

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses