Name: determining intestinal permeability using lucifer yellow in an apical-out enteroid model
Uploaded: 2022-07-27T14:35:00
Description: Watch this Scientific Journal Video about Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model at JoVE.com

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...

     

<ol>
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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 ...

Applying Lucifer Yellow to the media of Apical-out enteroids provides a simple method for quantitative and qualitative analysis of all three major paracellular intestinal permeability pathways in a 3D model. It is a relatively straightforward technique that allows us to assess permeability using an enteroid model. We are working with this technique in our studies of necrotizing enterocolitis.However, this technique can be useful in studying any human intestinal disease, utilizing an enteroid model. Demonstrating the procedures today will be our Post-Doctoral Research Scholars, Dr.Tyler Leiva, and Dr.Alena Golubkova, as well as Camille Schlegel, the Research Assistant Supervisor from our laboratory. To begin, grow the Androids embedded in BMM for seven to 10 days with 50%LWRN conditioned media for the apical-out or AO enteroid generation.Remove the media from around the BMM domes and add 500 microliters of cell recovery solution, or CRS to one well. Scrape the dome, pipette it up and down several times to homogenize the CRS with the enteroids embedded in BMM domes, and add this solution to the next well. Again, scrape the dome and pipette it up and down several times before transferring the solution to a microcentrifuge tube.Add 500 microliters of CRS to the second well, pipette up and down to mix it well and transfer it to the first well. From the first well, pool the CRS enteroid BMM solution into the same 1.5 milliliters microcentrifuge tube and repeat this pooling for the remaining wells until the contents of all the wells are in CRS. Place the microcentrifuge tubes containing pooled CRS solution on a rotator for one hour at four degrees Celsius.Then centrifuge the microcentrifuge tube at 200 X g for three minutes at four degrees Celsius before removing the supernatant, and re-suspending the enteroid pellet in one milliliter of 50%LWRN conditioned media. Gently transfer 500 microliters of the re-suspended enteroid or media solution to each well of the ultra-low attachment 24 well tissue culture plate. Incubate the plate at 37 degrees Celsius with 5%CO2, for three days prior to experimentation.Ensure that AO enteroids are suspended for at least 72 hours before use. Gently transfer all the enteroids suspended in media from each well into a microcentrifuge tube. Centrifuge at 300 X g for three minutes at room temperature and remove the supernatant.Add 10 microliters of five milligrams per milliliter of lipopolysaccharide, or LPS to 500 microliters of 50%LWRN conditioned media per well to prepare the master mix, and re-suspend the AO enteroid pellet from the treatment group into 500 microliters of LPS plus 50%LWRN conditioned media. Pipette up and down to thoroughly mix. Aliquot 500 microliters of suspension into each well of a 24 well ultra low attachment plate.Similarly re-suspend the AO enteroids from the untreated control group in 500 microliters of 50%LWRN conditioned media per well and aliquot 500 microliters of this suspension into each well of a separate 24 well ultra-low attachment plate. Induce hypoxia in the LPS treated AO enteroids using a modular incubator chamber with 1%oxygen, 5%carbon dioxide, and 94%nitrogen, and incubate the enteroids with hypoxia and LPS for 24 hours. Place the untreated and LPS plus hypoxia, treated cultures in a 5%carbon dioxide incubator for 24 hours.To measure the paracellular permeability using Lucifer Yellow or LY, gently transfer the enteroid or media suspension from each well into a microcentrifuge tube. Warm Dulbecco's phosphate buffered saline or DPBS, and 50%LWRN conditioned media in a 37 degrees Celsius water bath. Centrifuge the suspension at 300 X g for three minutes at room temperature.Remove the media, and wash the enteroid pellet with 500 microliters of warm DPBS before centrifugation at 300 X g for three minutes. Remove the supernatant with a micro pipette and repeat the DPBS wash step one more time. After the DPBS washes, add 450 microliters of warm 50%LWRN conditioned media, and add the suspension to a 24 well plate.Then add 50 microliters of 2.5 milligrams per milliliter LY to each well. Swirl the plate gently to mix, and place it in a 5%carbon dioxide incubator for two hours. Gently pipette the enteroid suspension from each well into a microcentrifuge tube.Centrifuge it and remove the supernatant leaving the enteroid pellet behind. Wash the pellet using 500 microliters of warm DPBS and centrifuge it. Remove the supernatant leaving the pellet behind.Repeat three washes of the DPBS before re-suspending the pellet with 1000 microliters of cold DPBS, and dissociate the enteroids by vigorous mixing using a pipette. For the Lucifer Yellow standard curve, prepare the standards diluted in DPBS as described in the text. Pipette 150 microliters of each sample per well in triplicate on a 96 well plate.Measure the fluorescence at an excitation peak of 428 nanometers and an emission peak of 536 nanometers on a fluorescence plate reader. Calculate the concentrations using statistical software and a four parameter curve interpolating the standard curve. Immunofluorescent staining of the enteroids suspended in 50%LWRN media for 72 hours showed Zo-1 on the outer apical surface of the enteroid, while beta catenin is seen on the basolateral surface.It indicated the expected polarity reversal of enteroids confirming an AO conformation of enteroids. Basolateral out, or BO enteroids treated with LPS and hypoxia showed significantly increased permeability which was demonstrated in a Lucifer Yellow, or LY assay by the increased uptake of LY dye into the enteroid lumen, compared to untreated controls. Lucifer Yellow assay also demonstrated the increased uptake of LY in the lumen of treated AO enteroids compared to untreated controls.It confirmed the increased permeability of AO enteroids treated with LPS and hypoxia, compared to the untreated controls. It's important to thoroughly wash the enteroid pellets to ensure that all the extraluminal LY is removed. It allows the accurate quantification of the remaining intraluminal LY in calculating the permeability.For qualitative analysis, enteroids can be visualized via fluorescent microscopy for LY uptake into the lumen. Following LY incubation and subsequent wash steps before dissociating from Microplate reading.

Research

JoVE Journal

Immunology and Infection

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

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

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A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

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

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

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