We thank Erika Mileti for excellent technical support and Dr. Antonio Di Sabatino for providing the oxygen chambers.
Funding: This work was supported by grants from the 7th EU framework program (IBDase, ERC: Dendroworld) and Fondazione Cariplo to MR and support by a Marie Curie international training mobility network (Cross-Talk, grant agreement No: 21553-2) to KT.

1. Obtaining and Preparing the Tissue
<ol>
	<li>Tissue (healthy or IBD mucosa) is obtained during surgery. Once the specimen is excised transfer to the lab as soon as possible, keeping it in Ca++/Mg++-free HBSS buffer supplemented with Pen/Strep at 4 &deg;C or on ice.</li>
</ol>
*The size of the specimen depends on the availability of the tissue: the part of the tissue obtained, is strictly what is not needed for diagnosis and can vary greatly between healthy and IBD subjects. Healthy tissue is excised from patients undergoing surgery for colon cancer.
<ol start="2">
	<li>Wash the tissue gently and clean it of all mesenteric material. Separate the mucosa layer from the submucosa using sterile scissors and forceps while keeping the tissue in HBSS buffer in a Petri dish (not necessarily immerged). Touch the mucosal surface with the forceps as little as possible.</li>
	<li>Cut mucosa in stripes at least 1 cm wide and as long as possible. Use two sterile scalpels, much as though you were cutting your food.</li>
	<li>Wash clean mucosa gently in HBSS and extend it on a Petri dish with the apical side facing upwards.</li>
</ol>
2. Mounting the Tissue
<ol>
	<li>Prepare fresh medium (tisDMEM). Use DMEM supplemented with Glutamine (2 mM) and NaPyr (1 mM), and add 15% FBS-Na (Fetal Bovine Serum - North American), 1% ITS-X and 200 ng/ml EGF at the time of use.</li>
	<li>Fill a 10 cm Petri dish with the rest of the FBS-Na (use about 30 ml so that metal grids will be completely submerged) and submerge the metal grids. Keep the plate open and support the plastic cylinders on its tap.</li>
</ol>
*The metal grids are made of iron. The mesh is square-shaped with a side of 0.5 mm.
<ol start="3">
	<li>Take 3 &mu;l of surgical glue with a 10 or 20 &mu;l pipette. Apply it carefully to one of the borders of a plastic cylinder while holding it firm with the forceps.</li>
	<li>Gently place the cylinder on the apical side of the mucosa, as close to one of the borders of the strip as possible.</li>
	<li>To give the glue time to firmly attach the cylinder on the tissue, prepare the centre-well organ culture plate: Dispense 850 &mu;l-1 ml of medium in the centre well and support the metal grid on the edges of the plate's central well.</li>
	<li>Cut away excess tissue from the borders of the cylinder using two sterile scalpels as before.</li>
	<li>Gently lift the cylinder with the attached tissue and place the basolateral side on the metal grid in the centre of the plate.</li>
</ol>
3. Stimulation
<ol>
	<li>Use the stimuli of your choice in your preferred concentration.</li>
	<li>Apply appropriate volume in the centre of the plastic cylinder without disturbing the cylinder or the mucosal surface with the pipette tip. Make sure your chosen volume uniformly covers the surface inside the cylinder. Indicated volumes: 
	Large plastic cylinder: 200 &mu;l 
	Medium plastic cylinder: 100 &mu;l 
	Small plastic cylinder: 50 &mu;l</li>
	<li>Incubate for up to 3 hr in a conventional incubator.</li>
</ol>
*If you wish to incubate for longer time points, make sure you use a much smaller volume of stimuli (10-20 &mu;l) and directly place the tissue in an atmosphere of 100% O2 in the pressure of 1 Atm (see 3.5).
<ol start="4">
	<li>Take the medium with the stimuli off the apical surface of the tissue and DO NOT REPLACE with fresh medium, because the tissue does not get enough O2 if a thick layer of medium on the apical face prevents gas exchange. Cover the plate.</li>
	<li>Place the tissue in an atmosphere of 100% O2 in the pressure of 1 Atm.</li>
</ol>
4. Harvesting
<ol>
	<li>After 24 hr of total culture time (&plusmn; 2.5 hr) carefully remove the cylinder from the tissue with the help of a scalpel gently scraping glue off and store it depending on the desired analysis (fix for immunohistochemistry and/or immunofluorescence assays, or snap freeze in liquid N2 for RNA purification and gene expression profiling or protein purification and Western blot assays).</li>
	<li>Harvest the basolateral medium for cytokine secretion profiling. Centrifuge at 8,000 rpm for 7 min to pellet debris, transfer the supernatant in an Eppendorf tube (alternatively aliquot) and immediately store at -20 &deg;C.</li>
</ol>
*if you want to keep supernatants for longer, store them at -80 &deg;C.

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

No conflicts of interest declared.

The need for physiologically relevant models on which treatments for human intestinal mucosa can be validly tested has long been emphasized. Many researchers have identified potential artifacts and defects in both cell 5 and mouse models 6 used so far for that purpose. Indeed, even though promising preclinical data is often obtained, these rarely translate to significant clinical benefit.
The method described in this work, presents a relatively simple, yet effective and novel adaptation to existing protocols 7, 8 for the culture and stimulation of human intestinal mucosa explants in a polarized fashion.
This model could provide an excellent complementary approach for the generation of valid pre-clinical data before taking a potential treatment of any kind to the clinics. Thus, unfortunate clinical outcomes 9 could potentially be avoided and researchers could more safely promote treatments to the clinics for the handling of acute inflammatory conditions.
The major advantages of the model are the possibility to compare polarized versus non polarized stimulation and to follow the tissue's response over a 24-hour time frame. A very important parameter is that in every experiment, the different treatments are applied on explants coming from the same patient which allows taking into account variability between patients. The fact that the explants are of a relatively large size compared to biopsies also means that a variety of analyses can be performed on the same explant, confirming results in many different ways (ELISA assays, quantitative rtPCRs, microarray analyses, immunohistochemistry and/or immunofluorescence data). Further, other types of analyses could be optimized in the future, such as purification of cell populations of interest from stimulated explants and evaluation of their phenotype by cytofluorimetric analysis.
However, there are also important limitations to be taken into account. At its present form this model is not suitable for high throughput screening of a large number of treatments, as it relies on the availability of the tissue. Cell culture models are probably more suitable for that purpose as they are easier to handle and perform a large number of stimulations on 10 whereas the model we describe would be more efficiently used as the end testing of candidate treatments before entering the clinics. Moreover, the time frame during which we have succeeded to preserve the viability of the tissue does not allow for experiments requiring long monitoring periods, such as RNA interference. Finally, the bacterial stimulation is carried out in a regular incubator and does not allow for preserving or testing the effect of anaerobic bacteria. It is our intention to work on these issues in the future.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>10X HBSS</td><td>Eurocalne</td><td>ECM4006XL</td><td></td></tr><tr><td>Peni/Strepto</td><td>Lonza</td><td>DE17602E</td><td></td></tr><tr><td>Gentamycin</td><td>Gibco</td><td>15750-037</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>BE12614</td><td></td></tr><tr><td>FBS-Na</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>100X ITS-X</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>EGF</td><td>Tebu-bio</td><td>100-15</td><td></td></tr><tr><td>L-Glutamine</td><td>Lonza</td><td>BE17605E</td><td></td></tr><tr><td>Non essential aminoacids 100X</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>NaPyr</td><td>Gibco</td><td>11360-039</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Cloning cylinders 6x8 mm</td><td>BellCo</td><td>2090-00608</td><td></td></tr><tr><td>Cloning cylinders 8x8 mm</td><td>BellCo</td><td>2090-0080</td><td></td></tr><tr><td>Cloning cylinders 10x10 mm</td><td>BellCo</td><td>2090-01010</td><td></td></tr><tr><td>Metal grids</td><td>Home made</td><td></td><td></td></tr><tr><td>Centre well organ culture plate</td><td>BD Falcon</td><td>353037</td><td></td></tr><tr><td>Surgical glue (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Oxygen Jars</td><td>Home made</td><td></td><td></td></tr><tr><td>Oxygen tanks</td><td>Air Liquid Sanit&amp;#224;</td><td></td><td></td></tr></tbody></table>

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 succeeded in maintaining healthy and IBD human intestinal mucosa in culture for at least 24 hr preserving the wellbeing of the tissue. In accordance with previous observations 1, this was only possible if the majority of the culture time (at least 85% of total) took place in O2, as the tissue did not survive for 24 hr in conventional incubators (Figure 2). We have also shown that different responses of the explants can be observed on this model depending on the polarity of the stimulation (apical vs basolateral) and the immunogenicity of the stimuli 2,3.
Using this protocol, we show that different probiotic strains can elicit different responses on the tissue's part. Lactobacillus plantarum NCIMB8826 proved to be surprisingly immunogenic on healthy tissue. Contrary to the two other Lactobacilli strains, not only did it induce migration of lymphoid cells towards the uppermost surface of the mucosa, but it also significantly upregulated a chemokine relevant to this migration, CCL4, as well as IL-1b, a cytokine traditionally related to pro-inflammatory activity. To our surprise, even though we had previously observed a preventive action of L. paracasei in a mouse model of colitis 4, this did not translate to a curative effect when live bacteria of this strain were applied on inflamed IBD tissue. Rather, all three strains proved detrimental when used on IBD mucosa in the acute phase of the disease 2.
Further, we showed that the metabolic product of a probiotic, called postbiotic, is capable of shielding healthy epithelium against a rather invasive Salmonella strain (FB62), while at the same time significantly ameliorates inflammation when applied on IBD mucosa 2.
<img alt="Figure 1" src="/files/ftp_upload/4368/4368fig1.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/4368/4368fig1hires.jpg"/> 
Figure 1. Schematic representation of the set-up and photos. a. schematic representation of the set up viewed from the side, b. schematic representation of the set up viewed from the top, c. photo of the metal grid used as support for the explant. When correctly placed, only the basolateral side gets in contact with the central medium chamber, d. photo of the entire ensemble. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4368/4368fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4368/4368fig2.jpg" /> 
Figure 2. The tissue only survives culture in O2. a. uncultured tissue, fixed upon arrival from the operating room, b. tissue cultured for 24 hr in O2, c. tissue cultured for 24 hr in a conventional incubator. Original magnification: 5X.
<img alt="Figure 3" src="/files/ftp_upload/4368/4368fig3.jpg" /> 
Figure 3. The effect of a pro-inflammatory stimulus on the explants. C-: Unstimulated tissue cultured for the indicated time points; Sal: Tissue challenged with Salmonella and cultured for the indicated time points. The destruction of the upper layer of the epithelium is already visible at 2 hr. The explant presents extensive degeneration after 24 hr. Original magnification: 10X.

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

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

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.

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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Nanyang Technological University

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JoVE Journal

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13.0K Views.  European Institute of Oncology. 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.

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

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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Immunology and Infection

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

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Medicine

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.

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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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Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

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Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

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3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

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 ...

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

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Chapters in this video

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

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

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

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

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

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