We thank Dr. Jerrold R. Turner, from Brigham and Women&#39;s Hospital, Harvard Medical School, for his generous help in completing this study. This work is supported by the National Natural Science Foundation of China (grant number&#160;81470804,&#160;31401229, and 81200620), the Natural Science Foundation of Jiangsu Province (grant number&#160;BK20180838, and BK20140319), The Research Innovation Program for College Graduates of Jiangsu Province (grant number KYLX16-0116), Advanced Research Projects of Soochow University (grant number SDY2015_06), and Crohn&#39;s &#38; Colitis Foundation Research Fellowship Award (grant number 310801).

This study was approved by the Animal Care and Use Protocol of Cambridge-Suda Genomic Resource Center (CAM-SU), Soochow University.
1. Plating and Maintenance of Caco-2bbe on Porous Polycarbonate Membranes
<ol>
	<li>Grow cells in a T75 flask with media (DMEM containing 10% FBS). Flasks should be fed regularly, depending on the cell density. 
	NOTE: For optimal plating, cells should divide rapidly and have a flat &#34;fried-egg&#34; shape, which indicates that cells are in the growth phase.</li>
	<li>Once cells are 80% confluent, take the flask out of the incubator and remove the media. Rinse any residual media with 1-2 mL of sterile PBS (without Ca2+). Pipet 1.5 mL Trypsin-EDTA into the flask and gently rock the flask; then, place the flask in the 37 &#176;C incubator for 20 min without rocking.</li>
	<li>While the cells are trypsinizing, place inserts containing porous polycarbonate membranes (pore size, 0.4 &#181;m; surface area, 0.33 cm2; see Table of Materials) into 24-well plates. Add 1.0 mL of culture media in the basal chamber (the lower space of the membrane).</li>
	<li>Pipet 5 mL of media into the flask and vigorously pipet the cells against the side of the flask 5-10 times to achieve loose, individual cells or 2-3 cell clumps.</li>
	<li>Plate 0.166 mL of cells (achieving a 1:8 dilution ratio) into the apical chamber (the upper space of the membrane). Incubate at 37 &#176;C for up to 3 weeks.
	<ol>
		<li>Feed cells three times weekly by carefully aspirating the media from the basal compartment of each well using a pressure pump. Gently drip 1 mL of media into the apical chamber of each insert.</li>
	</ol>
	</li>
</ol>
2. Use of Epithelial Meter (Volt/Ohm) for Measuring TER
NOTE: After about 3 weeks of culture on polycarbonate membranes, Caco-2BBe cells are ready for TER measurement.
<ol>
	<li>For cytokine studies, one day before measurement, replace the basal media with media containing 10 ng/mL of IFN&#947;. On the day of the experiment, replace the media with HBSS containing 2.5 or 7.5 ng/mL of TNF. 
	NOTE: IFN&#947; treatment increases expression of TNF receptor 2 (TNFR2)16.</li>
	<li>To correct the meter, insert the correction electrode into the Input port, and choose &#34;Ohm&#34; mode. Adjust the R Adj screw with a screwdriver until the meter displays a reading of 1,000 &#937;.</li>
	<li>Sterilize the electrodes by placing them in 70% ethanol for 15-30 min, and then allow them to air dry for 15 s. Rinse the electrode in the experimental cell culture media.</li>
	<li>Turn on the power, and choose the &#34;Ohm&#34; mode. Carefully place the long ends of the electrode bridges into the basal chamber and the short ends into the apical chamber. Ensure that the longer electrodes touch the bottom of the dish, while keeping the shorter electrodes below the surface of the media but above the tissue culture inserts. Keep the electrodes vertical.</li>
	<li>Measure the resistance of the sample inserts and blank inserts (i.e., the culture inserts without cells but with HBSS) at 0, 1, 2, 3, 4 h after cytokine treatment. Record the resistance.</li>
	<li>To achieve consistency across different plate formats, calculate the product of the resistance and the effective membrane area: 
	<img alt="Equation" src="/files/ftp_upload/57032/57032eq1.jpg" /> 
	For 24-well inserts, the effective membrane area is 0.33 cm2.</li>
</ol>
3. Murine Model of Dextran Sulphate Sodium (DSS)-induced Colitis
<ol>
	<li>Add DSS to autoclaved water to a final concentration of 3.5% (wt/vol).</li>
	<li>Administer 3.5% DSS to 8-week-old male C57BL/6 mice for a total of 7 days. Give regular drinking water without DSS to control mice.
	<ol>
		<li>Switch the DSS-containing water to regular drinking water after day 7.</li>
	</ol>
	</li>
	<li>Weigh mice and assess clinical scores of each mouse every day. Normalize body weight of each mouse to its initial body weight. Scores are defined according to the disease severity by four parameters: rectal prolapse (0-2), stool consistency (0-2), bleeding (0-2), and activity (0-2)5. Sum the scores from these parameters for a final clinical score.</li>
	<li>To analyze the histopathological condition of colon tissue, euthanize mice by intraperitoneal (i.p.) injection with 1.2% (vol/vol) Avertin (0.6 mL/10 g body weight) 7 days post-DSS treatment. To prepare a stock of 100% avertin, mix 10 g of 2,2,2-tribromoethanol with 10 ml of tert-amyl alcohol.&#160; Store in the dark at 4&#176;C. To use, dilute 100% stock to 2% in water.</li>
	<li>Isolate the colon and cecum, and measure the length of the colon5.</li>
	<li>Cut 0.5 cm segments from the distal colon and fix in a 15 mL falcon tube containing 10 mL of 10% formalin overnight. Wash the fixed tissues with graded ethanol (75, 95, and 100%) and xylene. Embed the tissues in paraffin and cut 6 mm sections for the hematoxylin &#38; eosin staining8.</li>
</ol>
4. Measuring the Epithelial Barrier Permeability in DSS-induced Colitis Mice
<ol>
	<li>Measure the barrier permeability 7 days after the start of DSS administration.</li>
	<li>On the day of the assay, fast mice for 3 h.</li>
	<li>Autoclave a gavage needle to ensure sterility, then gavage mice with 150 &#181;L 80 mg/mL 4 kDa FITC-dextran in sterile water, and keep the unused FITC-dextran to measure the standard curve after serum collection. Weigh the mice for the permeability calculation. 
	NOTE: The FITC-dextran solution should be made in water.</li>
	<li>Using a pair of scissors, clip a 1 cm piece of the tail, and collect 100 &#181;L of blood from the tail into serum collection tubes. Spin the collected blood at 10,000 x g for 10 min at room temperature.</li>
	<li>Dilute the serum 1:4 in water. To make a standard curve, dilute unused FITC-dextran with water at 1:300, 1:1,000, 1:3,000, 1:10,000, 1:30,000, 1:100,000, 1:300,000, 1:1,000,000, and 1:3,000,000. Add 100 &#181;L/well of the serum and the standard curve samples into 96-well plates.</li>
	<li>Read the fluorescence in a plate reader with 485 excitation/528 emission. Calculate the permeability values based on the standard curve, and multiply by 4 to correct for the dilution.</li>
	<li>Divide the concentration of FITC-dextran by the weight to normalize the values (this helps normalize the difference in FITC-dextran delivery if mice are sick and have lost weight).</li>
</ol>

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Biol. 36, 204-212 (2014).</li><li>Srinivasan, B., 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=TEER+measurement+techniques+for+in+vitro+barrier+model+systems.">TEER measurement techniques for in vitro barrier model systems.</a> J. Lab. Autom. 20 (2), 107-126 (2015).</li><li>Wang, F., 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131, 1153-1163 (2006).</li><li>Peterson, M. D., Mooseker, M. 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The authors declare no competing financial interests.

There are several critical steps in the protocol. Caco-2BBe (brush border-expressing) cells are always used for TER measurement, selected from the Caco-2 cell line for expression of brush-border proteins. Caco-2BBe cells have a villus absorptive phenotype when fully-differentiated (after about 3 weeks of culture post-confluence)17. It is necessary to avoid contamination during the measurement, and to sterilize the electrode. Because the procedure is non-sterile, measurements can only be performed for up to 8 h in most cases. During the measurement, we determined the resistance value of the two points inside and outside the insert. The resistance measurements at various sites can sometimes greatly differ because of differences in the cell state; This is a major limitation of the method. However, this variation can be significantly reduced by statistical methods and other methods. The TER method can be applied to other cells, such as determining the barrier integrity of pulmonary epithelial cells and vascular endothelial cells. In addition, TER measurements can be used in drug toxicity studies.
Administration of indigestible macromolecules, which enter the intestinal epithelium through diffusion, can be used to evaluate the intestinal permeability. The advantage of this method is that the barrier function of the intestine can be examined in vivo. There are several types of markers that can be used, including radioisotopes, polyethylene glycols, and sugars18. However, several factors should be considered for precise measurement of the permeability: (1) the position of gut barrier abnormality cannot always be precisely determined; and (2) the mucosal blood flow and gastrointestinal motility might affect the absorption and the excretion of the macromolecules.
In this study, 4 kDa FITC-dextran was administered intragastrically in mice and the amount of FITC-dextran in the mouse serum was measured. This method has numerous advantages. Oral gavage reduces possible risks that are associated with using enemas. For instance, unintentional impairment to the colonic epithelium may be caused by rectal instillation, leading to a false-positive result. Additional effects caused by stress should also be eliminated. Permeability is known to increase when the mice are stressed. It is recommended to conduct a preliminary experiment at least one week prior to the actual experiment. Performing a practice experiment will help minimize experimental noise for the real experiment. A wild-type control (no treatment) should always be included.
FITC-dextran travels through the small intestine and begins to enter the colon 3 h after gavaging. Thus, 3 h is an ideal time point for measuring small intestinal permeability. For measurement of colonic permeability, the time can be extended, but this then becomes a balance between the leakage of FITC-dextran into the lumen and clearance of these molecules from the blood stream. 4 h is generally accepted as a good time point for colonic measurements.
In conclusion, two separate methods were described to determine intestinal epithelial cell permeability both in vitro and in vivo. Whereas TER measurement and FITC-dextran gavage methods both measure paracellular permeability, they each provide independent and distinct properties of the paracellular permeability.

Intestinal epithelial cells are not only responsible for the absorption of nutrients, but also form an important barrier to defend against pathogenic microorganisms and microbial toxins. This intestinal barrier function is regulated by tight junction permeability and epithelial cell integrity1,2,3, and dysfunction of the epithelial barrier function is associated with inflammatory bowel disease (IBD). The perijunctional actomyosin ring (PAMR) lies within the cell that is closely contiguous to the tight junctions. The contraction of the PAMR, which is regulated by the myosin light chain (MLC), is crucial for the regulation of tight junction permeability4,5,6,7,8,9,10. Tumor necrosis factor (TNF) is central to intestinal barrier loss by upregulating intestinal epithelial MLC kinase (MLCK) expression and inducing occludin internalization11,12,13.
Ions such as Na+ and Cl- can cross the paracellular space by either the pore or leak pathway14. In a &#34;leaky&#34; epithelium, changes in TER primarily reflect altered tight junction permeability. TER measurement is a commonly used electrophysiological approach to quantify tight junction permeability, primarily to Na+ and Cl-, based on the impedance of cell monolayers. Diverse cell types, including intestinal epithelial cells, pulmonary epithelial cells, and vascular endothelial cells, have been reported for TER measurements. Advantages of this method are that TER measurements are non-invasive and can be used to monitor live cells in real-time. In addition, the TER measurement technique is useful for drug toxicity studies15.
Caco-2BBe cells are human epithelial colorectal adenocarcinoma cells with a structure and function similar to the differentiated small intestinal epithelial cells: for example, these cells have microvilli and enzymes associated with small intestinal brush border. Therefore, cultured Caco-2BBe monolayers are utilized as an in vitro model for testing barrier function.
In mice, one way to study intestinal paracellular permeability is by measuring the ability of FITC-dextran to cross from the lumen into the blood. Thus, the intestinal permeability can be assessed by gavaging FITC-dextran directly into mice and measuring the fluorescence within the blood. The following protocol describes two simple methods to assess intestinal epithelium permeability both in vitro and in vivo.

<table><tbody><tr><td>22 G gavage needle</td><td>VWR</td><td>20068-608</td><td></td></tr><tr><td>4 kDa FITC-dextran</td><td>Sigma</td><td>46944</td><td></td></tr><tr><td>Avertin</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Black 96-well plates for fluorescence</td><td>Fisher</td><td>14-245-197A</td><td></td></tr><tr><td>C57/B6 mice</td><td>Nanjing Biomedical Research Institute of Nanjing University</td><td></td><td></td></tr><tr><td>Caco-2BBe cells</td><td>ATCC</td><td>CRL-2102</td><td></td></tr><tr><td>Dextran sulphate sodium</td><td>MP Biomedicals</td><td>2160110</td><td></td></tr><tr><td>DMED with high glucose and sodium pyruvate</td><td>Hyclone</td><td>SH30243.01B</td><td></td></tr><tr><td>Epithelial (Volt/Ohm) Meter</td><td>Millicell-ERS</td><td>MERS00002</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm ChemicalReagent</td><td>10009218</td><td></td></tr><tr><td>Falcon tube (15 mL)</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Fluorometer</td><td>Biotek</td><td>Synergy 2</td><td></td></tr><tr><td>HBSS</td><td></td><td></td><td>138 mM NaCl, 0.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.4 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 0.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 5.0 mM KCl, 0.3 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 15.0 mM HEPES, 1.3 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 25 mM glucose&amp;nbsp;</td></tr><tr><td>IFNg</td><td>PeproTech</td><td>315-05-20</td><td></td></tr><tr><td>Modular Tissue Embedding Center</td><td>Leica</td><td>EG1150H</td><td></td></tr><tr><td>Serum collection tubes</td><td>Sarstedt</td><td>41.1378.005</td><td></td></tr><tr><td>T75 flask</td><td>corning</td><td>430641</td><td></td></tr><tr><td>TNF</td><td>PeproTech</td><td>315-01A</td><td></td></tr><tr><td>Parraffin</td><td>Sigma</td><td>A6330-1CS</td><td></td></tr><tr><td>Polycarbonate membranes (Transwell)</td><td>Costar</td><td>3413</td><td></td></tr><tr><td>Pressure pump</td><td>AUTOSCIENCE</td><td>AP-9925</td><td></td></tr><tr><td>Rotary Microtomy</td><td>Leica</td><td>RM2235</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Xylene</td><td>Sinopharm ChemicalReagent</td><td>10023418</td><td></td></tr></tbody></table>

in vitro and in vivo approaches to determine intestinal epithelial cell permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In culture, Caco-2BBe cells grow as a monolayer and slowly differentiate into mature absorptive enterocytes that have brush borders. In this protocol, Caco-2BBe cells were plated with a high density on polycarbonate membranes, and cells reached 100% confluency one day after seeding. However, cells are undifferentiated at this stage: To fully differentiate the cells, the media is changed every 2-3 days for 3 weeks. Cells were stained with nuclei and F-actin stains to show the differences between undifferentiated and differentiated cells. Stress fibers are clearly seen in undifferentiated cells. Compared with undifferentiated cells, differentiated cells have a smaller volume, larger nuclei, and fewer stress fibers (Figure 1).
TNF is central to intestinal barrier loss through MLCK-dependent tight junction regulation. TER of cells without or with TNF treatment at different time points was analyzed. As shown in Figure 2, TNF significantly decreases the TER of the Caco-2BBe monolayer in a dose-dependent manner, suggesting that TNF increases epithelial permeability.
Administration of DSS induces acute colitis in mice. Mice treated with DSS lose weight significantly compared to their initial body weight (Figure 3A). The severity of colitis is scored by rectal prolapse, stool consistency, bleeding, and activity (Figure 3B). Cross sections of colonic tissues stained with hematoxylin &#38; eosin show colonic mucosal damage in DSS treated mice (Figure 3C). The length of colon crypt is decreased in DSS treated mice (Figure 3D). The colon length is shortened in DSS treated mice (Figure 3E). As seen in Figure 3F, there is approximately a 2-fold increase in FITC-dextran levels in DSS treated mice compared to control mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57032/57032fig1.jpg" /> 
Figure 1: Caco-2BBe cells cultured on polycarbonate membranes. Cells cultured 1 day (undifferentiated) and 3 weeks (fully-differentiated) after plating are stained with Hoechst 33342 (blue) for the nucleus and Alexa Fluor 594 Phalloidin for F-actin (red). Scale bar = 20 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57032/57032fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57032/57032fig2.jpg" /> 
Figure 2: TNF reduces the TER of the Caco-2BBe monolayer. Cells are primed with 10 ng/mL of IFN&#947; overnight. On the next day, the culture medium is replaced with HBSS containing 2.5 or 7.5 ng/mL of TNF. TER is measured by an epithelial meter (Volt/Ohm) at the indicated time points. Values are mean &#177; SEM, n = 4. Significant differences are indicated by * (p &#60;0.05) and ** (p &#60;0.01), as compared to TER values of cells without TNF treatment, by two-tailed t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57032/57032fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57032/57032fig3.jpg" /> 
Figure 3: DSS-induced colitis in mice. C57BL/6 male mice are given 3.5% DSS in drinking water for 7 days. Body weight (A) and clinical scores (B) are assessed daily for each group (mean &#177; SEM, n = 4 for each group). Control mice received water without DSS. (C) Histological analysis of colonic tissue sections collected on day 7 post&#8211;DSS treatment. (D) Colon crypt lengths are measured on day 7 post&#8211;DSS treatment. (E) Colon lengths are measured on day 7 post&#8211;DSS treatment. (F) Permeability of colon epithelium is measured on day 7 post-DSS treatment by FITC-Dextran gavage. Values are mean &#177; SEM, n = 4. Significant differences are indicated by * (p &#60;0.05 by two-tailed t-test). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57032/57032fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Score</td>
			<td>Rectal prolapse</td>
			<td>Stool consistency</td>
			<td>Bleeding</td>
			<td>Activity</td>
		</tr>
		<tr>
			<td>0</td>
			<td>None</td>
			<td>Normal</td>
			<td>None</td>
			<td>Active</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Sign of prolapse</td>
			<td>Soft</td>
			<td>Red</td>
			<td>Decreased activity</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Extensive prolapse</td>
			<td>Diarrhea</td>
			<td>Gross bleeding</td>
			<td>Lethargic</td>
		</tr>
	</tbody>
</table>
Table 1. Disease Clinical Score

Watch this Scientific Journal Video about In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability at JoVE.com

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

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Nanyang Technological University

Research

JoVE Journal

Biology

25.2K Views.  Soochow University. Het algemene doel van dit experiment is om de ziekten te bestuderen die worden veroorzaakt door epitheliale barrièredisfunctie door de darmeppitheelceldoorlaatbaarheid in vitro en in vivo te meten. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het gebied van de darmbarrièrefunctie, zoals die met betrekking tot de inflammatoire darmziekte. Het belangrijkste voordeel van deze techniek is dat darmeppitheelceldoorlaatbaarheid zowel in vitro als in vivo kan worden beoord...

Video: In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Medicine

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunology and Infection

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

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