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>

<ol><li>Ranganathan, S., Smith, E. M., Foulke-Abel, J. D., Barry, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Research+in+a+time+of+enteroids+and+organoids%3A+How+the+human+gut+model+has+transformed+the+study+of+enteric+bacterial+pathogens%5BTitle%5D)+AND+%22Gut+Microbes%22%5BJournal%5D)">Research in a time of enteroids and organoids: How the human gut model has transformed the study of enteric bacterial pathogens.</a> Gut Microbes. 12 (1), 1795492(2020).</li>
<li>De Fazio, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Necrotizing+enterocolitis%3A+Overview+on+in+vitro+models%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Necrotizing enterocolitis: Overview on in vitro models.</a> International Journal of Molecular Sciences. 22 (13), 6761(2021).</li>
<li>Kovler, M. L., Sodhi, C. P., Hackam, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision-based+modeling+approaches+for+necrotizing+enterocolitis%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">Precision-based modeling approaches for necrotizing enterocolitis.</a> Disease Models & Mechanisms. 13 (6), (2020).</li>
<li>Ares, G. J., Buonpane, C., Yuan, C., Wood, D., Hunter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+human+epithelial+enteroid+model+of+necrotizing+enterocolitis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A novel human epithelial enteroid model of necrotizing enterocolitis.</a> Journal of Visualized Experiments. (146), e59194(2019).</li>
<li>Co, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlling+epithelial+polarity%3A+A+human+enteroid+model+for+host-pathogen+interactions%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Controlling epithelial polarity: A human enteroid model for host-pathogen interactions.</a> Cell Reports. 26 (9), 2509-2520 (2019).</li>
<li>Buonpane, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROCK1+inhibitor+stabilizes+E-cadherin+and+improves+barrier+function+in+experimental+necrotizing+enterocolitis%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology-Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">ROCK1 inhibitor stabilizes E-cadherin and improves barrier function in experimental necrotizing enterocolitis.</a> The American Journal of Physiology-Gastrointestinal and Liver Physiology. 318 (4), 781-792 (2020).</li>
<li>Hill, D. R., Huang, S., Tsai, Y. H., Spence, J. R., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+measurement+of+epithelial+barrier+permeability+in+human+intestinal+organoids%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Real-time measurement of epithelial barrier permeability in human intestinal organoids.</a> Journal of Visualized Experiments. (130), e56960(2017).</li>
<li>Lian, P., Braber, S., Varasteh, S., Wichers, H. J., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+and+heat+stress+affect+epithelial+integrity+in+a+Caco-2%2FHT-29+co-culture%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture.</a> Scientific Reports. 11, 13186(2021).</li>
<li>Zhao, W., Han, L., Bae, Y., Manickam, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lucifer+yellow+-+A+robust+paracellular+permeability+marker+in+a+cell+model+of+the+human+blood-brain+barrier%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Lucifer yellow - A robust paracellular permeability marker in a cell model of the human blood-brain barrier.</a> Journal of Visualized Experiments. (150), e58900(2019).</li>
<li>Manabe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chlorpheniramine+increases+paracellular+permeability+to+marker+fluorescein+lucifer+yellow+mediated+by+internalization+of+occludin+in+murine+colonic+epithelial+cells%5BTitle%5D)+AND+%22Biological+and+Pharmaceutical+Bulletin%22%5BJournal%5D)">Chlorpheniramine increases paracellular permeability to marker fluorescein lucifer yellow mediated by internalization of occludin in murine colonic epithelial cells.</a> Biological and Pharmaceutical Bulletin. 40 (8), 1299-1305 (2017).</li>
<li>Bardenbacher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Permeability+analyses+and+three+dimensional+imaging+of+interferon+gamma-induced+barrier+disintegration+in+intestinal+organoids%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids.</a> Stem Cell Research. 35, 101383(2019).</li>
<li>Miyoshi, H., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+expansion+and+genetic+modification+of+gastrointestinal+stem+cells+in+spheroid+culture%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.</a> Nature Protocols. 8 (12), 2471-2482 (2013).</li>
<li>Buonpane, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+modeling+of+necrotizing+enterocolitis+in+human+infant+intestinal+enteroids%5BTitle%5D)+AND+%22Journal+of+Investigative+Surgery%22%5BJournal%5D)">Experimental modeling of necrotizing enterocolitis in human infant intestinal enteroids.</a> Journal of Investigative Surgery. 35 (1), 111-118 (2022).</li>
<li>Chanez-Paredes, S. D., Abtahi, S., Kuo, W. -T., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiating+between+tight+junction-dependent+and+tight+junction-independent+intestinal+barrier+loss+in+vivo%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Differentiating between tight junction-dependent and tight junction-independent intestinal barrier loss in vivo.</a> Methods in Molecular Biology. 2367, 249-271 (2021).</li>
<li>Shen, L., Weber, C. R., Raleigh, D. R., Yu, D., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tight+junction+pore+and+leak+pathways%3A+A+dynamic+duo%5BTitle%5D)+AND+%22Annual+Review+of+Physiology%22%5BJournal%5D)">Tight junction pore and leak pathways: A dynamic duo.</a> Annual Review of Physiology. 73, 283-309 (2011).</li>
<li>Monaco, A., Ovryn, B., Axis, J., Amsler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epithelial+cell+leak+pathway%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">The epithelial cell leak pathway.</a> International Journal of Molecular Sciences. 22 (14), 7677(2021).</li>
<li>Srinivasan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TEER+measurement+techniques+for+in+vitro+barrier+model+systems%5BTitle%5D)+AND+%22Journal+of+Laboratory+Automation%22%5BJournal%5D)">TEER measurement techniques for in vitro barrier model systems.</a> Journal of Laboratory Automation. 20 (2), 107-126 (2015).</li>
<li>Kasendra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+primary+human+Small+Intestine-on-a-Chip+using+biopsy-derived+organoids%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids.</a> Scientific Reports. 8, 2871(2018).</li>
<li>Stroulios, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+methods+to+study+apical-specific+interactions+using+intestinal+organoid+models%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Culture methods to study apical-specific interactions using intestinal organoid models.</a> Journal of Visualized Experiments. (169), e62330(2021).</li>
<li>Frost, T. S., Jiang, L., Lynch, R. M., Zohar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Permeability+of+epithelial%2Fendothelial+barriers+in+transwells+and+microfluidic+bilayer+devices%5BTitle%5D)+AND+%22Micromachines%22%5BJournal%5D)">Permeability of epithelial/endothelial barriers in transwells and microfluidic bilayer devices.</a> Micromachines. 10 (8), 533(2019).</li>
</ol>

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><tbody><tr><td>[leu] 15-gastrin 1</td><td>Millipore Sigma</td><td>G9145-.1MG</td><td></td></tr><tr><td>100 &amp;micro;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.&lt;sup&gt;12&lt;/sup&gt;</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&amp;amp;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-&amp;beta;-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 ...

determining-intestinal-permeability-using-lucifer-yellow-an-apical

Research

JoVE Journal

Immunology and Infection

3.3K Views.  Oklahoma Children's Hospital. Het toepassen van Lucifer Yellow op de media van Apical-out enteroïden biedt een eenvoudige methode voor kwantitatieve en kwalitatieve analyse van alle drie de belangrijkste paracellulaire intestinale permeabiliteitsroutes in een 3D-model. Het is een relatief eenvoudige techniek die ons in staat stelt om de permeabiliteit te beoordelen met behulp van een enteroïde model. We werken met deze techniek in onze studies van necrotiserende enterocolitis.Deze techniek kan echter nuttig zijn ...

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

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

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Bioengineering

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. ...

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 ...

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 ...

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 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.

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

Biology

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 ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

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

Samenvatting

Meer video's verkennen