We would like to thank Ashley Nelson from the University of Rochester Medical Center for her instrumental help with our enteroid model. We would also like to thank the Division of Pediatric Surgery at the University of Oklahoma for their support of this project. This work was supported by the National Institute of Health [NIH Grant R03 DK117216-01A1], the Oklahoma Center for Adult Stem Cell Research, and the Presbyterian Health Foundation Grant #20180587 awarded to the Department of Surgery at the University of Oklahoma Health Sciences Center.

The present research was performed in compliance with Institutional Review Board approval (IRB, #11610, 11611) at the University of Oklahoma. Parental consent was required prior to collecting human surgical specimens as per IRB specifications. Following IRB approval and parental consent, human small intestinal tissue was obtained from infants (corrected gestational age (GA) ranging from 36-41 weeks at the time of sample collection, all with a history of preterm birth at an estimated GA of 25-34 weeks, 2:1 M:F) undergoing surgery for NEC or other intestinal resection, such as ostomy takedown or atresia repair. Enteroids were generated from tissue obtained from either the jejunum or ileum.
1. Human infant-derived enteroid cultures: crypt isolation and plating from whole tissue
<ol>
	<li>Prepare culture media, chelating buffer #1, and chelating buffer #2 (Table 1) following the previous report4. Store chelating buffers at 4 &#176;C and use them within 48 h.</li>
	<li>Prepare human minigut media (Table 1). Store the stock at &#8722;20 &#176;C and warm in a 37 &#176;C water bath prior to use.</li>
	<li>Prepare 50% L-WRN conditioned media (CM) (Table 1). Store the stock at 4 &#176;C for up to 1 week. 
	NOTE: 100% L-WRN base for the conditioned media is described in a protocol by Miyoshi et al.12.</li>
	<li>Generate enteroids from small intestinal tissue samples obtained from surgical specimens following the previous report4. Plate four wells of enteroids in a 24-well plate from a 0.75-2.5 g piece of intestinal tissue. 
	&#8203;NOTE: Enteroids can be successfully generated from tissue stored in 30 mL Roswell Park Memorial Institute (RPMI) 1640 medium in a 50 mL conical tube at 4 &#176;C for up to 48 h.</li>
	<li>Place the 24-well plate in a 37 &#176;C, 5% CO2 incubator upside-down for 15-20 min to allow for the polymerization of BMM domes4.</li>
	<li>Add 500 &#181;L of 50% L-WRN CM (as described in step 1.3) with 0.5 &#181;L of Y-27632, ROCK inhibitor (RI, see Table of Materials) to each well. After 2-3 days, replace with 50% L-WRN CM without RI.</li>
</ol>
2. Generation of AO enteroids
<ol>
	<li>Grow enteroids embedded in BMM for 7-10 days with 50% L-WRN CM4.</li>
	<li>Remove the media and add 500 &#181;L of cell recovery solution (CRS, see Table of Materials) to one well. Scrape the dome, pipette up and down several times, then add the CRS/enteroid/BMM solution to the next well.</li>
	<li>Scrape the dome, pipette up and down several times, and place the solution in a microcentrifuge tube. Add 500 &#181;L of CRS to the second well, pipette up and down, then add it to the first well. Transfer this solution to the same 1.5 mL microcentrifuge tube.</li>
	<li>Repeat step 2.3, pooling two wells into one microcentrifuge tube until all the well contents are in CRS. 
	NOTE: More than two wells can be pooled into one larger 15 mL conical tube if generating large volumes of AO enteroids. Maintaining a 500 &#181;L CRS per well ratio is important to ensure complete solubilization of BMM.</li>
	<li>Place the microcentrifuge tubes (step 2.3) with pooled CRS/enteroid/BMM solution on a rotator for 1 h at 4 &#176;C to solubilize the BMM.</li>
	<li>Centrifuge the solution at 200 x g for 3 min at 4&#176;C, then remove supernatant with a micropipette, leaving the pellet behind.</li>
	<li>Resuspend the enteroid pellet in 1 mL of 50% L-WRN CM (as prepared in step 1.3) per microcentrifuge tube. Gently pipette 500 &#181;L of the re-suspended enteroid/media solution into each well of the ultra-low attachment 24-well tissue culture plates (see Table of Materials).</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 3 days prior to experimentation.</li>
</ol>
3. Verification of AO enteroid conformation via whole-mount immunofluorescent staining
<ol>
	<li>Following incubation of the enteroids in media suspension for 3 days, pipette the enteroid/media suspension from one well into a microcentrifuge tube.</li>
	<li>Centrifuge the enteroid/media suspension at 200 x g for 5 min at 4 &#176;C. Remove the supernatant with a micropipette.</li>
	<li>Resuspend the enteroids in 20 &#181;L of BMM. Pipette 10 &#181;L of enteroid suspension onto a microscope slide and spread it in a thin layer approximately 1 cm by 1 cm square. Repeat with the remaining 10 &#181;L of enteroid suspension on a separate part of the same slide so that there are two smears of enteroid/BMM suspension.</li>
	<li>Allow the enteroid/BMM smears to solidify at room temperature (RT) for 15 min.</li>
	<li>Working in the fume hood, place the slide in a staining jar and fill with 4% paraformaldehyde until it covers the enteroid/BMM smear (~30 mL for one slide if using a glass staining jar with 10 slide capacity). Allow 30 min (at room temperature) for fixation to take place. 
	CAUTION: 4% paraformaldehyde is hazardous and may cause eye damage/irritation, skin irritation, and cancer. Always work under a fume hood when using it. Wear protective gloves and personal protective equipment when handling it. Dispose of it in an approved waste disposal container.</li>
	<li>Discard 4% paraformaldehyde in an appropriate waste container. Add 30 mL of phosphate-buffered saline (PBS) to the staining jar or until PBS covers the enteroid/BMM smears. Let it sit for 3 min at RT, and then discard the PBS in the paraformaldehyde waste bottle. Repeat this step two more times for a total of three washes.</li>
	<li>Fill a new staining jar with 30 mL of 0.5% Triton X-100 diluted in PBS. Place the slide with enteroid/BMM smears in the Triton solution and let it permeabilize for 20 min at RT.</li>
	<li>Discard the 0.5% Triton X-100 diluted in PBS. Add 30 mL of PBS to the staining jar and let it sit for 3 min. Discard the PBS by decanting.</li>
	<li>Gently wipe the slide around the enteroid/BMM smears, being careful not to disrupt them. Using a hydrophobic barrier pen (barrier PAP pen, see Table of Materials), draw a circle around each of the enteroid/BMM smears. Make a 20% serum, specific to the animal in which the secondary antibody was raised (see Table of Materials).</li>
	<li>Lay the microscope slide flat on the lab bench. Block the slide for 1 h at 4 &#176;C by pipetting 100 &#181;L of specific 20% serum from step 3.9 onto each of the enteroid/BMM smears ringed by the hydrophobic barrier pen.</li>
	<li>Prepare a 100 &#181;L solution with 1:100 and 1:200 dilutions of &#946;-catenin and ZO-1 primary antibodies, respectively, diluted in PBS. 
	NOTE: Each primary antibody must be from a different animal to ensure that they will have distinct immunofluorescent channels when imaged. The present study utilizes a mouse &#946;-catenin antibody and a rabbit ZO-1 antibody (see Table of Materials).</li>
	<li>Tap the slide on the counter to remove the 20% serum and gently wipe dry, being careful not to disrupt the enteroid/BMM smears. Pipette 100 &#181;L of &#946;-catenin/ZO-1 primary antibody solution onto one of the enteroid/BMM smears. Pipette 100 &#181;L of PBS without primary antibody onto the other enteroid/BMM smear as a negative control.</li>
	<li>Line the bottom of a plastic container with a wet paper towel to create a humidified container. Place the slide in the container, being careful not to disrupt solutions covering the enteroid/BMM smears. Place the lid on the container to seal, and store flat at 4 &#176;C overnight. 
	NOTE: Do not allow the slide to dry out. Ensure that the container is closed to avoid desiccation.</li>
	<li>Once ready to proceed, tap the slide on the counter to remove primary antibodies and PBS from the enteroid/BMM smears.</li>
	<li>Perform a wash step by placing the slide in a staining jar and filling with 30 mL of PBS or until PBS covers the enteroid/BMM smears. Let it sit for 3 min at room temperature. Discard the PBS and repeat this step twice more for a total of three washes.</li>
	<li>Prepare 200 &#181;L of secondary antibodies for two different immunofluorescent channels, with each antibody having a 1:1000 dilution in PBS (see Table of Materials). Keep the solution away from light.</li>
	<li>Pipette 100 &#181;L of secondary antibody solution onto each of the enteroid/BMM smears. Place it in the dark and let it sit for 1 h at RT.</li>
	<li>Tap the slide on the counter to remove secondary antibodies. Repeat the wash steps outlined in step 3.15, ensuring that all washes are performed in the dark.</li>
	<li>Add a drop of 4&#8242;,6-diamidino-2-phenylindole (Fluoroshield with DAPI, see Table of Materials) mounting medium to each of the enteroid/BMM smears and apply a coverslip to ensure that both smears are adequately covered. Avoid trapping bubbles under the coverslip. Keep the slide in the dark until ready to view under the microscope. 
	&#8203;NOTE: Immediate imaging of the slides yields the most optimal results; however, slides can be stored flat in a humidified container at 4 &#176;C and imaged for up to 1 week after the addition of DAPI.</li>
</ol>
4. Induction of experimental NEC
<ol>
	<li>Ensure that AO enteroids have been in suspension for at least 72 h prior to use.</li>
	<li>Gently pipette all the enteroid/media suspension from each well into a microcentrifuge tube. 
	NOTE: Multiple wells can be pooled into one 15 mL conical tube if a large volume of wells is treated. A minimum of three wells per treatment group is recommended for this protocol.</li>
	<li>Centrifuge at 300 x g for 3 min at RT and remove the supernatant.</li>
	<li>Add 10 &#181;L of 5 mg/mL of lipopolysaccharide (LPS, see Table of Materials) to 500 &#181;L of 50% LWRN CM per well (final concentration 100 &#181;g/mL LPS).</li>
	<li>Resuspend AO enteroids designated as the treatment group in 50% LWRN + LPS and aliquot 500 &#181;L of suspension to a 24-well ultra-low attachment plate. Resuspend AO enteroids designated as untreated controls in 500 &#181;L of 50% LWRN CM per well. Aliquot 500 &#181;L of this suspension to a separate 24-well ultra-low attachment plate.</li>
	<li>Induce hypoxia in the LPS-treated AO enteroids via a modular incubator chamber (MIC) with 1% O2, 5% CO2, and 94% N2 as per the manufacturer&#39;s instructions (see Table of Materials). Treat the enteroids with hypoxia and LPS for 24 h.</li>
	<li>Place the untreated and LPS + hypoxia-treated cultures, still in the MIC chamber, in a 37 &#176;C, 5% CO2 incubator for 24 h.</li>
</ol>
5. Measurement of paracellular permeability utilizing LY
<ol>
	<li>Gently pipette the enteroid/media suspension from each well into a microcentrifuge tube. Warm DPBS and 50% LRWN CM in a 37 &#176;C water bath.</li>
	<li>Centrifuge the enteroid/media suspension at 300 x g for 3 min at RT.</li>
	<li>Remove the media. Wash the enteroids with warm 500 &#181;L DPBS.</li>
	<li>Centrifuge at 300 x g for 3 min at RT. Remove the supernatant with a micropipette.</li>
	<li>Repeat steps 5.3-5.4 once more for a total of two times.</li>
	<li>After washing, add 450 &#181;L of warm 50% LWRN CM (as prepared in step 1.3).</li>
	<li>Add 50 &#181;L of 2.5 mg/mL LY (final concentration is 0.25 &#181;g/&#181;L, see Table of Materials) to each well and gently swirl to mix. Place in a 37 &#176;C, 5% CO2 incubator for 2 h.</li>
	<li>Gently pipette the enteroid/media suspension from each well into a microcentrifuge tube.</li>
	<li>Centrifuge at 300 x g for 3 min at RT, then remove the supernatant, leaving the enteroid pellet behind.</li>
	<li>Wash the enteroids with 500 &#181;L warm DPBS.</li>
	<li>Centrifuge at 300 x g for 3 min at RT, then remove the supernatant leaving the enteroid pellet behind.</li>
	<li>Repeat steps 5.10-5.11 three more times for a total of four washes.</li>
	<li>Resuspend each pellet with 1,000 &#181;L of cold DPBS and vigorously pipette up and down to dissociate the enteroids.</li>
	<li>Prepare standards for the LY standard curve diluted in DPBS (100 ng/mL; 50 ng/mL; 25 ng/mL; 12.5 ng/mL; 6.25 ng/mL; 3.13 ng/mL; 1.57 ng/mL; 0 ng/mL).</li>
	<li>Pipette 150 &#181;L of each sample per well in triplicate on a 96-well plate.</li>
	<li>Measure the fluorescence at an excitation peak of 428 nm and emission peak of 536 nm on a plate reader capable of reading fluorescence (see Table of Materials). Using statistical software (see Table of Materials), calculate the concentrations by interpolating the standard curve and using a four-parameter curve.</li>
</ol>

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The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.

Intestinal permeability is complex and reflective of epithelial barrier function. The intestinal barrier comprises a single layer of epithelial cells that mediates transcellular and paracellular transport14. Paracellular permeability relies on tight junction proteins that seal the space between epithelial cells14. Within this paracellular transport, there are three distinct pathways by which molecules can cross: pore, leak, and unrestricted15. The po...

Enteroids are three-dimensional (3D) structures derived from organ-restricted human intestinal stem cells1,2. They are made up entirely of epithelial lineage and contain all the differentiated intestinal epithelial cell types2. Enteroids also maintain cellular polarity made up of an apical luminal surface forming an inner compartment and a basolateral surface facing the surrounding media. Enteroids are a unique model in that they preserve the characteristics of the host from which they were generated3. Thus, enteroids generated from premature human infants represent a model that is useful for investigating diseases that primarily affect this population, such as necrotizing enterocolitis (NEC).
The traditional enteroid model is grown in a basolateral-out (BO) conformation, where the enteroid is encased in a dome of basement membrane matrix (BMM). BMM induces the enteroid to maintain a 3D structure with the basolateral surface on the outside. BO enteroids are a suitable model for NEC that bridges the gap between two-dimensional (2D) primary human cell lines and in vivo animal models2,4. NEC is induced in enteroids by placing pathogens such as LPS or bacteria in the media surrounding the enteroids, followed by exposure to hypoxic conditions2,3. The challenge with the BO enteroid NEC model is that it does not allow for the effective study of host-pathogen interactions, which occur at the apical surface in vivo. Changes in intestinal permeability are due to these host-pathogen interactions. To better understand how permeability affects the pathophysiologic basis of disease, a model must be created that involves treating the apical surface.
Co et al. were the first to demonstrate that mature BO enteroids can be induced to form an apical-out (AO) conformation by removing the BMM domes and resuspending them in media5. This article demonstrated that AO enteroids maintained correct epithelial polarity, contained all intestinal cell types, upheld the intestinal epithelial barrier, and allowed access to the apical surface5. Using AO enteroids as an NEC model achieves a physiological reproduction of the disease process and study of host-pathogen interactions.
One major contributor to the pathophysiology of NEC is increased intestinal permeability6. Several molecules have been proposed as a way to test for intestinal permeability in vitro7. Among these, lucifer yellow (LY) is a hydrophilic dye with excitation and emission peaks at 428 nm and 540 nm, respectively8. As it crosses through all the major paracellular pathways, it has been used to evaluate paracellular permeability in various applications, including the blood-brain and intestinal epithelial barriers8,9. The traditional application of LY uses cells grown in monolayers on a semi-permeable surface10. LY is applied to the apical surface and crosses through paracellular tight junction proteins to congregate on the basolateral side. Higher LY concentrations in the basolateral compartment indicate decreased tight junction proteins with subsequent intestinal epithelial cell barrier breakdown and increased permeability10. It has also been described in 3D BO enteroid models where LY was added to the media and individual enteroids were imaged for uptake of LY into the lumen11. Although this allows for qualitative analysis via the visualization of LY uptake, quantitative analysis is limited. This protocol outlines a unique technique that uses LY to assess paracellular permeability using an in vitro NEC enteroid model in AO enteroids while maintaining 3D orientation. This method can be used for both qualitative and quantitative analysis of permeability.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[leu] 15-gastrin 1</td>
			<td>Millipore Sigma</td>
			<td>G9145-.1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m sterile cell strainer</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>100% LWRN conditioned media</td>
			<td>Made in-house following Miyoshi et al.12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black, clear bottom plate</td>
			<td>Greiner Bio-One</td>
			<td>655090</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>R&#38;D Systems</td>
			<td>2939/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit secondary ab, 1:1000</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-mouse secondary ab, 1:1000</td>
			<td>Invitrogen</td>
			<td>A-11032</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-zonula occludens-1 rabbit primary ab, 1:200</td>
			<td>Cell Signaling</td>
			<td>#D6L1E</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-catenin mouse primary ab, 1:100</td>
			<td>Cell Signaling</td>
			<td>#14-2567-82</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement minus Vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier PAP pen</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>BMM (Matrigel)</td>
			<td>Corning</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354270</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-965-118</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320-082</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Millipore Sigma</td>
			<td>GF144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS m7000 Imaging system</td>
			<td>Invitrogen</td>
			<td>AMF7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio-Products</td>
			<td>100-525</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Millipore Sigma</td>
			<td>F6057-20mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 9</td>
			<td>Dotmatics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>12585014</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L2630-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Lucifer Yellow CH, Lithium Salt</td>
			<td>Invitrogen</td>
			<td>L453</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular incubator chamber</td>
			<td>Billups Rothenberg Inc.</td>
			<td>MIC101</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Millipore Sigma</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Millipore Sigma</td>
			<td>N0636-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated swinging bucket centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated tabletop microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Millipore Sigma</td>
			<td>S7067-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax iD3 microplate reader</td>
			<td>Molecular devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Revolver Rotator</td>
			<td>ThermoFisher Scientific</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low attachment 24-well tissue culture plate</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632, ROCK inhibitor (RI)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

AO conformation 
Enteroids suspended in 50% LWRN media for 72 h assume an AO conformation (Figure 1). This was confirmed via immunofluorescent staining utilizing enteroid whole mounts of the apical protein, zonula occludens-1 (ZO-1), and basolateral protein, &#946;-catenin (Figure 1). AO enteroids show ZO-1 (green) on the outer, apical surface of the enteroid, while &#946;-catenin (red) is on the inner, basolateral surface (<strong...

Watch this Scientific Journal Video about Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model at JoVE.com

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out 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 ...

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Research

JoVE Journal

Immunology and Infection

3.1K Views.  Oklahoma Children's Hospital. 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. 

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

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

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.

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 ...

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.

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 ...