Name: the ex vivo colon organ culture and its use in antimicrobial host defense studies
Uploaded: 2017-02-13T14:00:00
Description: Watch this Scientific Journal Video about The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies at JoVE.com

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
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<ol>
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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 pathophysiology...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/ F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, modified, w/o Calcium chloride &#38; 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&#160;</td>
			<td>BD Bioscience</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;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&#160;</td>
			<td>Bio-Rad</td>
			<td>1725125&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix S (1/8&#34;), 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>100x15 Petri Dish</td>
			<td>Falcon</td>
			<td>5687</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate 6well 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 micron 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 ...

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 overall goal of this procedure is to use ex vivo cultured mouse intestinal tissue to study the role of pathogen associated molecular patterns and the pattern recognition receptors for intestinal antimicrobial host defense responses.This method can help answer how innate immune pathways regulate intestinal microbiota via regulation of antimicrobial peptides.The main advantage of this technique is that the intestinal architecture remains intact while measuring antimicrobial peptides ex vivo.After euthanizing a mouse according to the text protocol, place the animal dorsal side down on a pinning board and pin the limbs.Then use 70%ethanol to spray the mouse.With presterilized dissecting scissors and forceps, make a midline incision in the peritoneum of the abdomen.Then use forceps to open the abdomen by folding the peritoneum.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.Place the colon in sterile Petri dish containing ice cold PBS, and then place it on a paper towel.Use the ice cold buffer and a 20 milliliter syringe, holding a 20g needle, or an oral gavage needle, to flush the contents of the lumen of the colon until the stool is completely removed.Using scissors, cut the colon longitudinally.Then wash the tissue by placing it into ice cold PBS in a sterile Petri dish and vigorously shaking.Repeat this wash three times.With a sterile scalpel or scissors, cut the colon tissue into pieces approximately one centimeter long, then record the weight of the colon pieces.To culture colon tissue, place a cell strainer on a six well cell culture plate.Then transfer all of the colon pieces collected from one mouse into the cell strainer.Add 5 milliliters of DMEM F12 medium containing 5%FBS, one x penicillin streptomycin, and 20 micrograms per milliliter of gentamycin, completely covering the colon pieces with the medium.Incubate the tissue at 37 degrees Celsius with 5%CO2 and 95%air for two hours.Then lift the cell strainer and aspirate the medium.Next, place the cell strainer back on the well and add five milliliters of fresh medium without antibiotics.Then lift the cell strainer and aspirate the medium.Repeat the wash two more times to remove all of the residual antibiotics.Transfer the colon pieces from a single cell strainer into a single well of a sterile 12 well cell culture plate.Add one milliliter of DMEM F12 medium containing 5%FBS per one milligram of tissue, then incubate the tissue at 37 degrees Celsius, injecting 5%CO2 and 95%air for 12 hours.Following the incubation, collect the culture supernatant in a sterile 1.5 milliliter tube.Centrifuge the sample at 12, 000 times g and four degrees Celsius for five minutes.Separate the supernatant into a new 1.5 milliliter tube for the use in the bacteria killing assay and/or other immune assays.Store the supernatant at minus 80 degrees Celsius until ready to perform the assay.To carry out an e.coli killing assay, inoculate e.coli into give milliliters of LB in a 15 milliliter tube.While keeping the cap on the culture tube slightly loose incubate the cell culture at 37 degrees C while shaking at 200 rpm overnight.Centrifuge the bacterial culture tube at 12, 000 times g and four degrees Celsius for 10 minutes.Then remove the supernatant and resuspend the bacterial pellet in five milliliters of ice cold PBS.Transfer one milliliter of the bacterial suspension into a cuvette and measure the optical density at 600 nanometers.Use a predetermined standard curve to calculate the colony forming units.In this experiment, assume that one optical density unit equals two times 10 to the ninth CFU per milliliter.Next, dilute the bacterial suspension to make a stock suspension of one times 10 to the fifth colony forming units per milliliter.Then transfer the previously collected colon organ culture supernatant and control media into duplicate wells of a 24 well cell culture plate.Add 10 microliters of e.coli culture into one well of the supernatant and control.Leave the duplicate wells without e.coli to confirm the colon organ culture supernatant is not contaminated.Incubate the culture at 37 degrees Celsius for one hour.After collecting colons from mice as demonstrated earlier in this video, place the colon on a sterile paper towel and use scissors to cut the colon longitudinally into three parts.Use ice cold PBS to wash each part of the colon.Then cut each part of the organ into small pieces and weigh.Transfer the colon pieces into three separate cell strainers placed on three wells of a six well cell culture plate.Add five milliliters of DMEM F12 medium containing 5%FBS, one x penicillin streptomycin, and 20 micrograms per milliliter gentamycin.Incubate the tissue at 37 degrees Celsius and 5%CO2 for two hours.After washing the colon pieces as demonstrated earlier in this video, transfer the tissue from a single cell strainer into a single well of a sterile 12 well cell culture plate.Designate the three wells as untreated, IL-1

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Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...