This work was supported by funding from Crohn&#39;s and Colitis Foundation of America, (CCFA; 3711) Cancer Prevention and Research Institute of Texas (CPRIT; RP160169), and UT Southwestern Medical Center given to M.H.Z.

All experiments described here were performed using 6-8 weeks old male wild-type (C57BL6/J) mice maintained in a specific pathogen free (SPF) facility at the Animal Resource Center (ARC), UT Southwestern Medical Center. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the IACUC guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Collection and Preparation of the Colon
<ol>
	<li>Euthanize mice with CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Spray mice with 70% ethanol and pin limbs to a pinning board keeping the mouse dorsal side down on the board.</li>
	<li>Using pre-sterilized dissecting scissors and forceps, make a mid-line incision in the peritoneum of the abdomen. Open the abdomen by folding the peritoneum with forceps.</li>
	<li>Remove the intestine from the abdominal cavity with dissecting forceps and scissors. Separate the colon from the small intestine by cutting at the bottom of the cecum and the other end at the rectum.</li>
	<li>Place the colon in a sterile petri dish containing ice-cold PBS. Flush the contents of the lumen of the colon with ice-cold PBS using a 20 mL syringe holding a 20 G needle or an oral gavage needle until the stool within the lumen of the colon is completely removed.</li>
	<li>Cut the colon with scissors longitudinally. Wash the colon by shaking vigorously in ice-cold PBS in a sterile Petri dish.</li>
	<li>Cut the colon tissue into pieces approximately 1 cm long using a sterile scalpel or scissors.</li>
	<li>Record the weight of the colon pieces. 
	NOTE: All the steps in the Section 1 can be performed either inside or outside of a biological safety cabinet. Once colons are ready for culture, all the steps must be performed inside a biological safety cabinet to maintain sterility.</li>
</ol>
2. Colon Organ Culture
<ol>
	<li>Place a cell strainer (100 &#956;m) on a 6-well cell culture plate. Transfer all of the colon pieces collected from one mouse into the cell strainer.</li>
	<li>Add 5 mL DMEM/F12 medium containing 5% FBS, Penicillin-Streptomycin (1x), and Gentamycin (20 &#181;g/ml). Completely cover the colon pieces with the media.</li>
	<li>Incubate for 2 hr at 37 &#176;C in an incubator with 5% CO2 and 95% air.</li>
	<li>Lift the cell strainer and aspirate the media.</li>
	<li>Place the cell strainer back on the well and add 5 ml fresh media without any antibiotics. Lift the cell strainer and aspirate the media.</li>
	<li>Repeat the above washing step (Step 2.5) twice more (total 3x) to remove all of the residual antibiotics.</li>
	<li>Transfer the colon pieces from a single cell strainer into a single well of a sterile 12-well cell culture plate.</li>
	<li>Add DMEM/F12 medium containing 5% FBS (without any antibiotics). Adjust the volume of the medium with the weight of the colon pieces, e.g., 1 ml medium for 100 mg of tissue. Incubate for 12 h at 37 &#176;C in an incubator injecting 5% CO2 and 95% air.</li>
	<li>Collect the culture supernatant in a sterile 1.5 ml tube.</li>
	<li>Centrifuge at 12,000 x g at 4 &#176;C for 5 min. Separate the supernatant into a new 1.5 mL tube for use in the bacteria killing assay and/or other immune assays. The supernatant can be stored at -80 &#176;C until the assay.</li>
	<li>Collect the colon pieces in a tube for RNA isolation.</li>
</ol>
3. E. coli Killing Assay
<ol>
	<li>Inoculate E. coli in 5 mL Luria-Bertani (LB) broth in a 15 ml tube and incubate at 37 &#176;C with shaking at 200 rpm overnight. Keep the cap of the culture tube slightly loose.</li>
	<li>Centrifuge the bacterial culture tube at 1,200 x g for 10 min at 4 &#176;C. Remove the supernatant and resuspend the bacterial pellet into 5 ml ice-cold PBS.</li>
	<li>Transfer 1 mL of the bacterial suspension into a cuvette and measure OD at 600 nm. Use PBS as a blank.</li>
	<li>Calculate the colony forming unit (cfu) using a predetermined standard curve. Here, assume that 1 OD = 2 x 109 cfu/ml (approximately).</li>
	<li>Dilute the bacterial suspension to make the stock suspension 1 x 105 cfu/mL.</li>
	<li>Transfer the colon organ culture supernatant (500 &#181;l/well) collected in Section 2.10 into duplicate wells of a 24-well cell culture plate.</li>
	<li>Add 10 &#181;L E. coli culture (1,000 cfu) into one well containing 500 &#181;L colon organ culture supernatant. Leave the other well without any E. coli inoculation to confirm that the colon organ culture supernatant does not contain any contamination.</li>
	<li>Incubate the same number of bacteria (1,000 cfu) in the culture media (DMEM/F12 plus 5% FBS) without any antibiotics as a control.</li>
	<li>Incubate at 37 &#176;C for 1 h.</li>
	<li>Place 50 &#181;L of each sample drop-wise on MacConkey agar plates. Incubate the MacConkey agar plates at 37 &#176;C overnight.</li>
	<li>Count the number of colonies and calculate the cfu/ml.</li>
</ol>
4. The Effect of Extrinsic and Intrinsic Factors on Colonic Antimicrobial Host Defense Responses
NOTE: The antimicrobial killing assay as described here can be adopted to examine the effect of pathogen associated molecular patterns (PAMPs) and cytokines on bactericidal activity of organ culture supernatant. An example of such experiment using IL-1&#946; and IL-18 is described below.
<ol>
	<li>Collect colons from mice as described in Section 1.</li>
	<li>Place the colon on a sterile paper towel. Cut the colon longitudinally into three parts using scissors (Figure 2).</li>
	<li>Wash each part of the colon in ice cold PBS as described in Section 1.</li>
	<li>Cut each part of the colon into small pieces and weigh aseptically. Transfer the pieces of each part of the colon into three separate cell strainers (100 &#956;m) placed on three wells of a 6-well cell culture plate (Figure 2).</li>
	<li>Add 2 ml DMEM/F12 medium containing 5% FBS, Penicillin-Streptomycin (1x), and Gentamycin (20 &#181;g/ml).</li>
	<li>Incubate for 2 h at 37 &#176;C in an incubator injecting 5% CO2 and 95% air.</li>
	<li>Wash the colon pieces as described in 2.4-2.6. Transfer the colon pieces of a single cell strainer into a single well of a sterile 12-well cell culture plate. The three wells containing three parts of the colon from a single mouse should be designated as untreated, IL-1&#946;, and IL-18 (Figure 2).</li>
	<li>Add DMEM/F12 medium containing 5% FBS (without any antibiotics). Adjust the volume of the medium with the weight of the colon pieces, e.g., 1 mL medium for 100 mg of tissue.</li>
	<li>Stimulate the colon organ culture with IL-1&#946; (20 ng/mL) or IL-18 (20 ng/mL) for 12 h. The untreated colon organ culture serves as control.</li>
	<li>After 12 h incubation at 37 &#176;C in an incubator injecting 5% CO2 and 95% air, collect the culture supernatant in a sterile 1.5 mL tube.</li>
	<li>Transfer 500 &#181;L culture supernatant into duplicate wells of a 24-well plate.</li>
	<li>Inoculate E. coli (1,000 cfu) in colon organ culture supernatant as described in Section 3. For each condition, inoculate E. coli in a single well. The other well containing same organ culture supernatant without E. coli will serve as a control for bacterial contamination.</li>
	<li>Incubate the same amount of bacteria in culture media without any antibiotics as a control.</li>
	<li>Incubate at 37 &#176;C for 1 h.</li>
	<li>Place 50 &#181;L of each sample drop-wise on MacConkey agar plates. Incubate the MacConkey agar plates for overnight at 37 &#176;C.</li>
	<li>Count the number of colonies and calculate the cfu/mL (Figure 4).</li>
</ol>
5. Measurement of the Expression of Antimicrobial Genes
<ol>
	<li>After overnight incubation (12 h) of colonic organ culture as described in Sections 2 and 4, wash the colon pieces with 2 ml ice-cold PBS (2x).</li>
	<li>Collect the colon pieces into a 2 mL RNase/DNase free screw-cap tube. Place the tubes on ice.</li>
	<li>Add 1 mL commercial Trizol reagent and lysing matrix beads into the tube.</li>
	<li>Lyse the tissue using an automated tissue homogenizer.</li>
	<li>Collect the tissue lysate into a new microcentrifuge tube.</li>
	<li>Isolate RNA using standard protocol.</li>
	<li>Measure RNA concentration.</li>
	<li>Dilute RNA appropriately with deionized water and use 500 ng RNA to synthesize cDNA.</li>
	<li>Use the cDNA for real-time RT-PCR analysis of targeted antimicrobial genes, cytokines, chemokines, and other genes of interest.</li>
</ol>

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The authors have no competing financial interests.

The intestinal epithelial cells are very sensitive in terms of their growth requirements and therefore difficult to culture. The epithelial cells isolated by EDTA treatment do not survive in conventional cell culture media such as DMEM8. Therefore, host-pathogen interaction studies using isolated crypt or primary epithelial cells are very challenging. Recently, Sato et al. described a crypt organoid culture system which is very promising and useful for studies related to intestinal pathophysiology13. While organoids represent many features of intestinal crypt, there are concerns as to whether the immune responses of the organoid cells, which are being grown in a sterile environment, recapitulate the responses of intestinal epithelial cells in vivo. The precise techniques required for isolation and culture of epithelial stem cells and requirement of expensive reagents for the culture of organoids prevent many laboratories from taking advantage of this system. While the use of ex vivo organ culture is not an alternative to organoid culture, this system offers an easy and inexpensive method to study the antimicrobial responses of intestinal epithelium.
The major advantage of this technique is that the tissue architecture of the intestine is maintained while culturing in the dishes. We observed that the colon tissues are physiologically active for at least 24 h after their culture. During the culture of the colon, the colonic epithelial cells respond to exogenous stimuli as measured by expression of cytokines and antimicrobial peptides. The viability of the epithelial cells of the intact tissue can be further enhanced by the addition of appropriate growth factors and inhibitors of cell death in the culture medium. Previous reports suggest that the normal human colonic tissue organ could be maintained ex vivo for several days8,23,24.
The protocol for the ex vivo culture of mouse colonic organ may have many applications in studies related to antimicrobial immune responses of the intestinal epithelium. The pathogens and their PAMPs are recognized by PRRs such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) present in epithelial and immune cells. Defective expression and function of many TLRs and NLRs are associated with susceptibility to IBD, colorectal tumorigenesis, and bacterial infections. Since immortalized epithelial cell lines do not represent all the features of the intestinal niche, the ex vivo colon organ culture is a very useful experimental tool. Using this system, we can examine the effect of various PAMPs or stimuli on the induction of antimicrobial effector molecules in the intestinal epithelial cells. In the representative experiment, we showed that cytokines like IL-1&#946; and IL-18 trigger the expression of antimicrobial peptides (Figure 3). We also show here that antimicrobial peptides produced by colon tissue can effectively kill E. coli (Figure 4). These techniques can be applied to test the influence of lipopolysaccharide (LPS), peptidoglycan (PGN), muramyldipeptide (MDP), and other PAMPs in antimicrobial host defense responses in the gut.
We have slightly modified the colon organ culture protocol as described previously12,16,20,25. We cultured the colon in two phases. At first, we incubated the colon tissue in antibiotics containing medium for 2 h. We then removed the antibiotics with repeated washing of the colon pieces followed by incubation with antibiotics free media. Thus, the culture supernatant obtained from overnight culture of the colon exhibits only the effect of secreted antimicrobial peptides when incubated with E. coli. This approach can be utilized to see the bactericidal effect of colon or intestine derived antimicrobial peptides on any bacteria of interest. An appropriate selective agar media should be used to culture the bacteria.
Like most techniques, there are limitations of the ex vivo organ culture system. First, unlike cell culture or organoid culture, the organs do not grow and proliferate and therefore cannot be expanded. Second, intestinal epithelial cells are very sensitive and they die relatively quickly without additional growth factors and apoptosis inhibitors, which are used for crypt organoid culture. Third, the immune response observed from the organ culture is a collective response of different kinds of cell populations. Therefore, the role of individual cell type in the expression of antimicrobial and effector genes cannot be assessed. Additionally, unlike in cell lines and organoid culture, the expression of antimicrobial peptides and other effector molecules in the colons may vary from mouse to mouse of same genetic background.
In summary, we describe here a simple protocol for ex vivo colonic organ culture that is very useful for studying intestinal antimicrobial immune responses. This approach can be employed to test the role of diverse innate immune genes in the expression of antimicrobial peptides by culturing colons from genetic mutant mice of interest. Further, this protocol can be adapted for use in clinical studies to examine the intestinal antimicrobial responses of IBD patients by conducting organ culture of colonic biopsy samples.

The intestine represents a dynamic system that acts as a barrier for commensal microorganisms, fights against invading pathogens, and regulates the microbial composition1. The intestinal epithelial cells, consisting of enterocytes, goblet cells, Paneth cells and enteroendocrine cells, are the major cell populations that provide host defense responses against intestinal microbiota. The goblet cells produce mucins that create a demilitarized zone on the top of the epithelial layer2. The Paneth cells and enterocytes produce antimicrobial peptides, cytokines, and reactive oxygen and nitrogen species that constitute antimicrobial host defense responses and contribute to shaping the intestinal microbial composition3,4. In addition to epithelial cells, the immune cells including macrophages, dendritic cells, neutrophils, natural killer cells, lymphocytes, and innate lymphoid cells in the lamina propria and submucosa play a critical role in intestinal antimicrobial host defense responses by producing cytokines, chemokines, and other mediators5-7. In order to understand how the mucosal immune system regulates microbiota and provides protection against microbial infection, it is important to consider the complex interaction of the heterogeneous cell populations of the gut. However, an in vitro model that encompasses all of the features of the intestine is not available. Therefore, molecular studies on host-pathogen interaction in the intestine are highly challenging.
Over the past few years, several model systems that mimic aspects of the intestinal mucosa have been developed for investigating the pathophysiological processes involved in inflammatory bowel diseases (IBD) and other gastrointestinal disorders8-14. Immortalized intestinal epithelial cell lines are often used to study epithelial cell specific responses. However, because of differential gene expression and function in immortalized cells, the data obtained from using those cells do not often match with those observed in in vivo studies. Intestinal crypt organoid culture has recently emerged as a potential tool for assessing the response of the intestinal epithelium to different stimuli13. In this system, crypt stem cells are allowed to grow and develop a 3D organoid structure. While the organoid culture system is very useful for studying many aspects of the intestinal epithelium, it does not mimic the complex interaction of immune cells, epithelial cells and microbial products. The ex vivo culture of the intestinal tissue offers a better representation of in vivo host defense responses. In this method, a part of the intestine is cultured in a cell culture plate with appropriate media allowing the different types of cell populations in the intestine to be metabolically active for at least 48 h. Thus, an ex vivo culture of the organ can be used to measure the expression of antimicrobial genes and the host defense responses of the intestine to a particular stimulus.
Investigators have been using the ex vivo organ culture system to study host defense responses against microbial infection in the intestine15-21. We recently adopted the organ culture system to study the role of the inflammasome in antimicrobial host defense responses in mouse colons22. The inflammasome is a molecular platform for the activation of caspase-1, which is required for the production of matured IL-1&#946; and IL-18. We showed that IL-1&#946; and IL-18 induce antimicrobial peptides which effectively kill commensal pathobionts such as E. coli. This observation was consistent with increased E. coli burden in inflammasome-defective mouse colons22. This system therefore can be used to study the role of pattern recognition receptors (PRRs) and other innate immune molecules in intestinal antimicrobial host defense responses as well as pathogenesis of intestinal disorders such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). There are more than 200 IBD susceptibility genes, and mutations in many of these genes are associated with altered microbial composition in the gut. It is of great clinical significance to determine the precise mechanism through which the IBD-susceptibility genes regulate gut microbiota. The overall goal of this method is to introduce a basic protocol of ex vivo colon organ culture and demonstrate how this culture method can be used to study antimicrobial host defense responses of the intestine.

<table><tbody><tr><td>Advanced DMEM/ F12</td><td>Life Technologies</td><td>12634-010</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, modified, w/o Calcium chloride &amp;amp; Magnesium chloride</td><td>Sigma</td><td>5634</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1272</td><td></td></tr><tr><td>Mouse IL-1b recombinan</td><td>Reprokine</td><td>RKP10749</td><td></td></tr><tr><td>Mouse IL-18 recombinant</td><td>Reprokine</td><td>RKP70380</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Difco Luria-Bertani Broth&amp;nbsp;</td><td>BD Bioscience</td><td>244620</td><td></td></tr><tr><td>BD&amp;nbsp;Difco Dehydrated Culture Media: MacConkey Agar</td><td>Fisher Scientific</td><td>DF0075-17-1</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>Uded to measure RNA concentration</td></tr><tr><td>UV/Vis Spectrophotometer</td><td>BECKMAN</td><td>DU 530</td><td>Used to determine E. coli count</td></tr><tr><td>iScript RT Supermix, 100 rxns</td><td>Bio-Rad</td><td>1708841</td><td></td></tr><tr><td>iTaq Univer SYBR Green Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>1725125&amp;nbsp;</td><td></td></tr><tr><td>Lysing Matrix S (1/8&quot;), 2 ml Tube</td><td>MP Biomedicals</td><td>116925500</td><td>Used to homgenize colon organ for RNA isolation</td></tr><tr><td>FastPrep-24 5G System</td><td>Bio-Rad</td><td>116005500</td><td></td></tr><tr><td>100 mm x 15 mm Petri Dish</td><td>Falcon</td><td>5687</td><td></td></tr><tr><td>Plate 6 well ps TC CS100, Cellstar, 6w, tc, F-bottom (Flat), w/lid, sterile</td><td>Cellstar</td><td>5085</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Falcon</td><td>5698</td><td></td></tr><tr><td>Sorvall Legend Micro 21R Centrifuge</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sorvall ST40R Centrifuge</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Forma Scientific orbital shaker</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr></tbody></table>

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.

A representative picture of colons in organ culture is shown in Figure 1. The colon pieces in the culture remain metabolically and physiologically active. They respond efficiently to exogenous stimuli added to the culture media. A schematic work flow of the preparation of the colon tissue for ex vivo culture and stimulation with exogenous stimuli, e.g. IL-1&#946; and IL-18, is shown in Figure 2. The representative data in Figure 3 show that colons in organ culture express antimicrobial peptides such as &#946;-defensin 2 (BD2), Reg3&#947;, S100a8, S100a9, and iNOS in response to IL-1&#946; and IL-18.
The mediators released by the colon implants exhibit an antimicrobial killing effect as demonstrated by the significantly reduced growth of E. coli incubated with the organ culture supernatant (Figures 4A and 4B). Such E. coli killing activity is further potentiated by the stimulation of IL-1&#946; and IL-18 (Figures 4A and 4B). Colon organ culture supernatants incubated without E. coli show no bacterial growth following culture on MacConkey agar (Figures 4C and 4D). These results further suggest that the inflammasome plays an important role in the intestinal antimicrobial host defense.
Overall, the data presented here suggest that ex vivo colonic organ culture is a very useful technique to study intestinal antimicrobial immune responses in vitro.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GAPDH_F</td>
			<td>&#160;TGGCAAAGTGGAGATTGTTGCC</td>
		</tr>
		<tr>
			<td>GAPDH_R&#160;</td>
			<td>AAGATGGTGATGGGCTTCCCG</td>
		</tr>
		<tr>
			<td>&#946;-defensin 2 (BD2)_F</td>
			<td>&#160;TGACCACTGCCACACCAATG</td>
		</tr>
		<tr>
			<td>&#946;-defensin 2 (BD2) _R&#160;</td>
			<td>CCTGGCAGAAGGAGGACAAA</td>
		</tr>
		<tr>
			<td>Reg3&#947;_F</td>
			<td>&#160;CAAGGTGAAGTTGCCAAGAA</td>
		</tr>
		<tr>
			<td>Reg3&#947;_R&#160;</td>
			<td>CCTCTGTTGGGTTCATAGCC</td>
		</tr>
		<tr>
			<td>S100a8_F</td>
			<td>&#160;TGTCCTCAGTTTGTGCAGAATATAAA</td>
		</tr>
		<tr>
			<td>S100a8_R&#160;</td>
			<td>TCACCATCGCAAGGAACTCC</td>
		</tr>
		<tr>
			<td>S100a9_F&#160;</td>
			<td>GGTGGAAGCACAGTTGGCA</td>
		</tr>
		<tr>
			<td>S100a9_R&#160;</td>
			<td>GTGTCCAGGTCCTCCATGATG</td>
		</tr>
		<tr>
			<td>iNOS_F</td>
			<td>TGTGACACACAGCGCTACAACA</td>
		</tr>
		<tr>
			<td>iNOS_R</td>
			<td>GAAACTATGGAGCACAGCCACAT</td>
		</tr>
	</tbody>
</table>
Table 1: List of mouse primers for real-time PCR.
<img alt="Figure 1" src="/files/ftp_upload/55347/55347fig1.jpg" /> 
Figure 1: Representative picture of mouse colon pieces in the culture. (A) Incubation of colon pieces on a cell strainer (100 &#956;m) placed on a 6-well plate. Colon pieces are submerged in 2 mL culture media supplemented with antibiotics. (B) Following 2 h incubation, colon pieces are transferred into the well of a new 12-well cell culture plate containing antibiotics-free medium. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55347/55347fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55347/55347fig2.jpg" /> 
Figure 2: Schematic presentation of the steps for the preparation of colon tissues and ex vivo stimulation with IL-1&#946; and IL-18. The colon from one mouse is longitudinally dissected into three parts. Each part was cut into small pieces and weighed. The colon pieces from each section of the colon are transferred onto cell strainers placed on wells of a 6-well plate containing 2 ml DMEM/F12 medium plus 5% FBS and antibiotics. Following a 2 h incubation, colon pieces from a single cell strainer are transferred into a single well of a 12-well cell culture plate. DMEM/F12 plus 5% FBS (no antibiotics) is added into each well and the volume of the medium is adjusted according to the weight of the tissue (1 mL media for 100 mg tissue). Colon tissues are stimulated with IL-1&#946; (20 ng/ml) and IL-18 (20 ng/ml) for 12 h. Culture supernatants are centrifuged and used for E. coli killing assay and/or other immune assays. The colon tissues are collected into commercial trypsin reagent for RNA isolation and subsequent analyses of antimicrobial gene expression by real-time PCR. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55347/55347fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55347/55347fig3.jpg" /> 
Figure 3: Mouse colon tissues express cytokines and antimicrobial peptides in response to IL-1&#946; or IL-18 during ex vivo culture. Colon organ cultures were stimulated with IL-1&#946; (20 ng/ml) or IL-18 (20 ng/ml) for 12 h. The expression of BD2, Reg3&#947;, S100a8, S100a9, and iNOS was measured by real-time RT-PCR (Table 1). Data represent means &#177; SD; *p &#60;0.05, **p &#60;0.01. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55347/55347fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55347/55347fig4.jpg" /> 
Figure 4: Supernatant of mouse colon organ culture exhibits bactericidal activity. Colon pieces were cultured ex vivo in the presence or absence of IL-1&#946; (20 ng/mL) or IL-18 (20 ng/mL) for 12 h. 500 &#181;l of the culture supernatants were incubated with E. coli (1,000 cfu) for 1 hr at 37 &#176;C. Colon organ culture supernatant without E. coli was also incubated as a control. Following 1 h incubation, 50 &#181;L of each sample was placed dropwise on MacConkey agar plates and incubated at 37 &#176;C overnight. (A) Picture of E. coli colonies on MacConkey agar showing antimicrobial activity of colon organ culture. (B) E. coli count showing killing efficiency of IL-1&#946;- and IL-18-treated colon organ culture supernatant. (C) Picture of MacConkey agar plate showing no bacterial growth in colon organ culture incubated without E. coli. (D) No E. coli colonies were identified in colon organ culture incubated without E. coli, indicating the absence of any contaminating bacteria in the sample. Data represent means &#177; SD; **p &#60;0.01, ***p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55347/55347fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies at JoVE.com

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 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 ...

the-ex-vivo-colon-organ-culture-its-use-antimicrobial-host-defense

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.0K Views.  UT Southwestern Medical Center. 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. 

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

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

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Biochemistry

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Medicine

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

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.

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Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

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A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

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Biology

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.

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

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Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

An Ex Vivo Gut Organ Culture System to Study Host-Microbiota Interactions

Source: Azriel, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62779">An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.</a> J. Vis. Exp. (2021).
In this video, we describe the methodology for setting up an ex vivo gut organ culture system to study the interactions between mouse intestinal tissue fragments and specific gut bacteria. The device consists of two pairs of input-output ports, the first connecting the intestinal tissue lumen and supplying the specific bacterial suspension into it, and the second continuously supplying media to the device&#39;s wells to maintain ex vivo tissue viability.

System hodowli narządów jelitowych ex vivo do badania interakcji między gospodarzem a mikrobiotą

Source: Azriel, S., et al. An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions. J. Vis. Exp. (2021).
In this video, we ...

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

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System hodowli narządów jelitowych ex vivo do badania interakcji między gospodarzem a mikrobiotą