We thank Dr. Ian Glass and the personnel at the Birth Defects Research Laboratory at the University of Washington for sharing the fetal tissues. We also thank Dr. Michael Dame and Dr. Jason Spence at the Translational Tissue Modeling Laboratory at the University of Michigan for their endless support and guidance throughout the process.

<ol><li>Humberg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preterm+birth+and+sustained+inflammation%3A+Consequences+for+the+neonate%5BTitle%5D)+AND+%22Seminars+in+Immunopathology%22%5BJournal%5D)">Preterm birth and sustained inflammation: Consequences for the neonate.</a> Seminars in Immunopathology. 42 (4), 451-468 (2020).</li>
<li>Kim, J., Koo, B. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+organoids%3A+Model+systems+for+human+biology+and+medicine%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Human organoids: Model systems for human biology and medicine.</a> Nature Reviews Molecular Cell Biology. 21 (10), 571-584 (2020).</li>
<li>Pinto, D., Gregorieff, A., Begthel, H., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Canonical+Wnt+signals+are+essential+for+homeostasis+of+the+intestinal+epithelium%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.</a> Genes & Development. 17 (14), 1709-1713 (2003).</li>
<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449 (7165), 1003-1007 (2007).</li>
<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li>
<li>Mahe, M. M., Sundaram, N., Watson, C. L., Shroyer, N. F., Helmrath, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+human+epithelial+enteroids+and+colonoids+from+whole+tissue+and+biopsy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Establishment of human epithelial enteroids and colonoids from whole tissue and biopsy.</a> Journal of Visualized Experiments. (97), e52483(2015).</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>Stewart, C. J., Estes, M. K., Ramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+human+intestinal+enteroid%2Forganoid+lines+from+preterm+infant+and+adult+tissue%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Establishing human intestinal enteroid/organoid lines from preterm infant and adult tissue.</a> Methods in Molecular Biology. 2121, 185-198 (2020).</li>
<li>Finkbeiner, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome-wide+analysis+reveals+hallmarks+of+human+intestine+development+and+maturation+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Transcriptome-wide analysis reveals hallmarks of human intestine development and maturation in vitro and in vivo.</a> Stem Cell Reports. 4 (6), 1140-1155 (2015).</li>
<li>Senger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+fetal-derived+enterospheres+provide+insights+on+intestinal+development+and+a+novel+model+to+study+necrotizing+enterocolitis+%28NEC%29%5BTitle%5D)+AND+%22Cellular+and+Molecular+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Human fetal-derived enterospheres provide insights on intestinal development and a novel model to study necrotizing enterocolitis (NEC).</a> Cellular and Molecular Gastroenterology and Hepatology. 5 (4), 549-568 (2018).</li>
<li>Alganabi, M., Lee, C., Bindi, E., Li, B., Pierro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+understanding+necrotizing+enterocolitis%5BTitle%5D)+AND+%22F1000+Research%22%5BJournal%5D)">Recent advances in understanding necrotizing enterocolitis.</a> F1000 Research. 8, (2019).</li>
<li>Dame, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+colonic+crypts+in+culture%3A+Segregation+of+immunochemical+markers+in+normal+versus+adenoma-derived%5BTitle%5D)+AND+%22Laboratory+Investigation%22%5BJournal%5D)">Human colonic crypts in culture: Segregation of immunochemical markers in normal versus adenoma-derived.</a> Laboratory Investigation. 94 (2), 222-234 (2014).</li>
<li>Hill, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+colonization+stimulates+a+complex+physiological+response+in+the+immature+human+intestinal+epithelium%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium.</a> eLife. 6, 29132(2017).</li>
<li>Tsai, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+cryogenic+preservation+of+human+biopsy+specimens+and+subsequent+organoid+culture%5BTitle%5D)+AND+%22Cellular+and+Molecular+Gastroenterology+and+Hepatology%22%5BJournal%5D)">A method for cryogenic preservation of human biopsy specimens and subsequent organoid culture.</a> Cellular and Molecular Gastroenterology and Hepatology. 6 (2), 218-222 (2018).</li>
<li>Su, K., Asbrock, N., Chu, V., Hoffmann, S., Hewitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vitro+Differentiation+of+Human+iPS+Cells+Into+Colon+Organoids+in+Serum-Free+Cell+Culture+Conditions%5BTitle%5D)+AND+%22Technology+Networks%22%5BJournal%5D)">In Vitro Differentiation of Human iPS Cells Into Colon Organoids in Serum-Free Cell Culture Conditions.</a> Technology Networks. , https://www.technologynetworks.com/cell-science/white-papers/in-vitro-differentiation-of-human-ips-cells-into-colon-organoids-in-serum-free-cell-culture-328450 (2019).</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>Schoultz, I., Keita, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Intestinal+Barrier+and+Current+Techniques+for+the+Assessment+of+Gut+Permeability%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">The Intestinal Barrier and Current Techniques for the Assessment of Gut Permeability.</a> Cells. 9 (8), 1909(2020).</li>
<li>Kumar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+lineage-specific+transcription+factor+CDX2+navigates+dynamic+chromatin+to+control+distinct+stages+of+intestine+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The lineage-specific transcription factor CDX2 navigates dynamic chromatin to control distinct stages of intestine development.</a> Development. 146 (5), (2019).</li>
<li>Walker, R. W., Clemente, J. C., Peter, I., Loos, R. J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prenatal+gut+microbiome%3A+Are+we+colonized+with+bacteria+in+utero%5BTitle%5D)+AND+%22Pediatric+Obesity%22%5BJournal%5D)">The prenatal gut microbiome: Are we colonized with bacteria in utero.</a> Pediatric Obesity. 12, Suppl 1 3-17 (2017).</li>
<li>Spence, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470 (7332), 105-109 (2011).</li>
<li>Shin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+gradient+and+instability+of+Wnt+induce+heterogeneous+growth+and+differentiation+of+human+intestinal+organoids%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Spatiotemporal gradient and instability of Wnt induce heterogeneous growth and differentiation of human intestinal organoids.</a> iScience. 23 (8), 101372(2020).</li>
<li>Lueschow, S. R., McElroy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Paneth+cell%3A+The+curator+and+defender+of+the+immature+small+intestine%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">The Paneth cell: The curator and defender of the immature small intestine.</a> Frontiers in Immunology. 11, 587(2020).</li>
<li>In, J. G., Foulke-Abel, J., Clarke, E., Kovbasnjuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+colonoid+monolayers+to+study+interactions+between+pathogens%2C+commensals%2C+and+host+intestinal+epithelium%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Human colonoid monolayers to study interactions between pathogens, commensals, and host intestinal epithelium.</a> Journal of Visualized Experiments. (146), e59357(2019).</li>
<li>Dheer, R., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem-cell-derived+models%3A+Tools+for+studying+role+of+microbiota+in+intestinal+homeostasis+and+disease%5BTitle%5D)+AND+%22Current+Opinion+in+Gastroenterology%22%5BJournal%5D)">Stem-cell-derived models: Tools for studying role of microbiota in intestinal homeostasis and disease.</a> Current Opinion in Gastroenterology. 37 (1), 15-22 (2021).</li>
</ol>

All authors declare that they have no conflicts of interest to disclose.

This protocol details the establishment of enteroids from fetal intestinal tissue, as well as model characterization with immunofluorescent staining and epithelial permeability testing. The permeability of the enteroids was tested using a microinjection technique and serial time course measurements of leaked dextran-FITC concentration in the culture media. The novelty of this protocol is the apical exposure that more closely resembles human intestinal physiology compared to basolateral exposure in the culture media7. In previous studies, Hill et al. utilized serial imaging and calculation of the fluorescence intensity over time16. Ares et al. exposed the basolateral membrane of an epithelial model to LPS and then compared the cellular gene expression pattern7. In comparison, we use apical exposure of the tested reagents and then examine potential alterations in gross permeability by measuring serial concentrations of leaked dextran in the culture media. Our method also allows for serial comparative analysis of cytokines produced by epithelial cells in culture media and gene expression by collecting cellular mRNA. LPS has been commonly used to study intestinal injury in animal and in vitro models because of its ability to induce permeability and inflammation7,17. When LPS was tested in this model, epithelial permeability was differentiated by exposure concentration. This protocol can be expanded to study other disease pathologies using different microinjected materials and outcome measurements.
Critical steps in this protocol include establishing enteroids from fetal intestinal tissues, enteroid characterization, and the microinjection technique. The integrity of this study depends on accurate cell sampling. Using anatomical landmarks and blood vessels is helpful to ensure the selection of small intestinal cells. Due to the robust growth and differentiation of fetal intestinal stem cells, an extensive procedure to isolate the stem cells from other epithelial cells is not necessary. After the enteroids have been established, it is important to confirm the characteristics of the model by staining for proteins and cell markers. In this protocol, the enteroids were stained for enterocytes using villin and CDX2, Paneth cells using lysozyme, and goblet cells using mucin, all the cells which are found within the small intestine epithelium18,19,20. In contrast to traditional single cell lines that display one cell type, enteroids establish all cell types from the intestinal progenitor stem cells8. The staining portion of this protocol can be modified for the specific cellular markers of interest. Crucial to the reliability of this protocol is the microinjection technique. Consistency of the micropipette tips can be verified by measuring the volume per pump using dextran-FITC solution and visualizing the diameter of the tips under the microscope. Due to the risk of contamination, the same micropipette cannot be used for more than one exposure. Additionally, the shape and growth of the enteroids can be influenced by the contents of their growth media. We found that the spherical rather than cauliflower-shaped enteroids provided better models for microinjection. The spherical shape may be induced by a greater Wnt factor in the media21.
This protocol largely depends on the performer&#39;s skill in microinjection to reduce variations, especially in time-sensitive measurements. Variations can be minimized by having the same experienced performer with consistent techniques, using the same cell origin to avoid genetic variation, testing at the same passage to remove maturity bias, and growing the cells in the same type of media for similar cell differentiation. The components of the growth media may induce varying differentiation of stem cells in vitro rather than in an in vivo environment. For example, in vivo, lysozyme is not expressed until weeks 22-24 of gestational development when Paneth cells form and become functional22. However, we were able to detect lysozyme in our enteroids established from 10-week fetal intestines. This method can limit the number of tested exposures at one time due to the high technical skill required for microinjection. The dextran leakage from the microinjection puncture hole can affect the assessment of permeability. To eliminate this effect, triple washes immediately after the microinjection are recommended to remove residual dextran from the procedure. The dextran concentration in the media should be measured hourly for 4-6 h post microinjection. Experiments with a significant rise in dextran concentration within 2-4 h post injection should be excluded from the final analysis.
This method has several advantages. It requires a lower cost and fewer resources in comparison to enteroid-derived monolayers on transwell. Additionally, it can be expanded to other exposures such as live bacteria or viruses to study the initial interaction between gut microbes and the epithelium. The enclosed lumen of the enteroid can maintain a stable growth of microinjected live bacteria without contamination of the growth media13. Unlike the monolayer being exposed to growth media and incubation oxygen, the enclosed lumen is a tight, isolated space. An enclosed lumen allows for no communication to the growth media and a luminal oxygen content that is lower over time with live bacterial growth13.
The use of fetal intestinal tissue more accurately depicts the intestinal epithelium of preterm infants as compared to adult intestinal stem cells or animal models7. Further, the polarity of enteroids allows for both apical and basolateral exposures and measurements23. The enteroids form an enclosed lumen with lower oxygenation concentration, which more closely mimics the oxygenation concentration of the intestines24. In contrast to more technologically advanced protocols16, the use of gross media measurements allows for greater accessibility of this technique. The experiment demonstrated that epithelial leakage can be induced by apical exposure to LPS and is concentration-dependent. Since this method examines the changes in leaked concentration of dextran, it is useful for detecting gross functional changes in tight junctions. Messenger RNA collection and sequencing of the exposed enteroids and western blot analysis can complement analysis of the gross functional changes. This model studies intestinal epithelium integrity in a system that highly resembles the preterm environment and, thus, can be used to gain a better understanding of preterm intestinal injuries and other disease pathologies.

Preterm infants are exposed to frequent and prolonged inflammation that puts them at an increased risk for intestinal injury resulting in long-term disability or death1. Research in this area is challenged by the limited ability to conduct experiments on vulnerable preterm infants. Moreover, a lack of suitable models has hampered the comprehensive study of the premature intestinal environment2. Existing in vitro and in vivo models have failed to comprehensively represent the premature human intestinal environment. Specifically, single epithelial fetal cell lines may not form tight junctions, and animal models exhibit different inflammatory and immunological responses than human preterm infants. With the discovery of Wnt signaling as a primary pathway in the proliferation and differentiation of intestinal crypt stem cells and the novel Lgr5+ tissue stem cells, intestinal tissue-derived organoids such as enteroids and colonoids were established as in vitro models3,4,5. Using this technology, it is possible to create and utilize three-dimensional (3D) enteroid models that are developed from whole tissue or biopsy of an intestine to study epithelial responses to the intestinal environment6,7.
In contrast to typical intestinal cell lines grown in culture, enteroids exhibit polarity with a lumen connected by tight junction proteins8. This allows for exposure to the basolateral border in the growth media, as well as luminal microinjection to assess the apical border. Further, enteroids display similar genetic, physiologic, and immunologic characteristics as the human epithelium9,10. Fetal tissue-derived enteroids allow for the examination of the role of prematurity on epithelial function. The unique characteristics of enteroids can resemble the preterm intestinal environment more closely9. Tissue-derived enteroids can be used to test for tight junction integrity as a monolayer form or as 3D structures embedded in a solidified basement membrane protein mixture. A microinjection technique is required for the latter form if an apical exposure is desired. The measurement of epithelial responses in enteroid models includes gene expression by RNA sequencing, biomarkers by enzyme-linked immunoassay (ELISA), or advanced imaging techniques. The technique presented here provides another feasible option for measuring gross permeability with fluorometry.
Intestinal injury in preterm infants has a multifactorial pathogenesis that includes the imbalance of the gut microbial community. Enteroids can provide excellent models to study certain aspects of preterm intestinal diseases such as necrotizing enterocolitis that involve epithelial functions11. Enteroids display similar characteristics as the human fetal intestine10. Exposing enteroids to lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, in the culture media as a basolateral exposure induces gene expression that can lead to increased inflammation and intestinal permeability7. This study aims to evaluate the changes in gross epithelial permeability after apical exposure to bacterial products such as LPS. The results may provide insight into the microbe-epithelial interactions involved in the pathogenesis of intestinal injury. The method designed to test gross permeability requires microinjection setup and skill.

<table><tbody><tr><td>Amphotericin 250 uL/mL</td><td>Gibco</td><td>15-290-026</td><td></td></tr><tr><td>Anti-CDX-2 [CDX2-88] 0.5mL concentrated Mouse, IgG, monoclonal</td><td>Biogenex</td><td>MU392A-5UC</td><td></td></tr><tr><td>Anti-Claudin 2 antibody (ab53032)</td><td>abcam</td><td>ab53032</td><td></td></tr><tr><td>Anti-Claudin 3 antibody (ab15102)</td><td>abcam</td><td>ab15102</td><td></td></tr><tr><td>Anti-LYZ antibody produced in rabbit</td><td>Millipore Sigma</td><td>HPA066182-100UL</td><td></td></tr><tr><td>Anti-Mucin 2/MUC2 Antibody (F-2): sc-515032</td><td>Santa Cruz</td><td>sc-515032</td><td></td></tr><tr><td>Anti-Villin, Clone VIL1/4107R 0.5mL concentrated Rabbit, IgG, monoclonal</td><td>Biogenex</td><td>NUA42-5UC</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Millipore Sigma</td><td>A8806</td><td></td></tr><tr><td>Cell culture plates, CytoOne 12-well non-treated plates&amp;nbsp;</td><td>USA Scientific Inc</td><td>50-754-1395</td><td></td></tr><tr><td>Cell culture plates, CytoOne 6-well non-treated plates&amp;nbsp;</td><td>USA Scientific Inc</td><td>50-754-1560</td><td></td></tr><tr><td>Centrifuge with 15 mL tube buckets</td><td>Eppendorf&amp;nbsp;</td><td>05-413-110</td><td></td></tr><tr><td>CHIR 99021</td><td>Tocris Bioscience</td><td>4423</td><td></td></tr><tr><td>Confocal microscope&amp;nbsp;</td><td>Olympus FV1200</td><td>N/A</td><td>Or a similar microscope</td></tr><tr><td>Conical centrifuge tubes, 15 ml</td><td>Falcon</td><td>05-527-90</td><td></td></tr><tr><td>Cover glass for microscope slides</td><td>Fisher Scientific</td><td>12-544-DP</td><td></td></tr><tr><td>Disposable scalpels</td><td>Mopec</td><td>22-444-272</td><td></td></tr><tr><td>Dmidino-2-phenylindole (DAPI) Solution (1 mg/mL)</td><td>Fisher Scientific</td><td>62248</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-buffered Saline (DPBS, 1X)</td><td>Fisher Scientific</td><td>AAJ67802K2</td><td></td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid tetrasodium salt (EGTA)</td><td>Millipore Sigma</td><td>E8145-10G</td><td></td></tr><tr><td>Fluorescein isothiocyanate dextran (Dextran-FITC) 4 kDa</td><td>Millipore Sigma</td><td>46944</td><td></td></tr><tr><td>Fuorescence microplate reader</td><td>Agilent BioTek</td><td>Synergy HTX</td><td></td></tr><tr><td>Gentamicin 50 mg/mL</td><td>Gibco</td><td>15-750-060</td><td></td></tr><tr><td>Glass capillary tubes, single-barrel borosilicate, 1x0.5mm, 6&quot; (cut in half before pulling)</td><td>A-M systems</td><td>626500</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594</td><td>Fisher Scientific</td><td>A32742</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td><td>Fisher Scientific</td><td>A32731</td><td></td></tr><tr><td>Goat Serum</td><td>Fisher Scientific</td><td>16210064</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td>N/A</td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>IntestiCult Organoid Growth Medium (Human)</td><td>Stem Cell&amp;nbsp; Technologies</td><td>&amp;nbsp;06010</td><td></td></tr><tr><td>Lipopolysaccharides from&amp;nbsp;Escherichia coli&amp;nbsp;O111:B4</td><td>Millipore Sigma</td><td>L4391-1MG</td><td></td></tr><tr><td>Magnetic stand</td><td>World Precision Instruments&amp;nbsp;</td><td>M10</td><td></td></tr><tr><td>Matrigel Basement Membrane Matrix, LDEV-free, 10 mL&amp;nbsp;</td><td>Corning</td><td>354234</td><td>&amp;nbsp;protein concentration &gt; 9 mg/mL preferably</td></tr><tr><td>Micro forceps</td><td>Fisher Scientific</td><td>13-820-078</td><td></td></tr><tr><td>Micro scissors</td><td>Fisher Scientific</td><td>08-953-1B</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments&amp;nbsp;</td><td>M3301</td><td></td></tr><tr><td>Micropipette puller</td><td>World Precision Instruments&amp;nbsp;</td><td>SU-P1000</td><td>Or a similar equipment</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>22-034486</td><td></td></tr><tr><td>Occludin Polyclonal Antibody</td><td>Fisher Scientific</td><td>&amp;nbsp;71-1500</td><td></td></tr><tr><td>Paraformaldehyde 32% aqueous solution</td><td>ELECTRON&amp;nbsp;MICROSCOPY SCIENCES</td><td>RT 15714</td><td></td></tr><tr><td>Petri Dishes, 35x10 mm</td><td>Fisher Scientific</td><td>150318</td><td></td></tr><tr><td>Petri Dishes, 60x15 mm</td><td>Fisher Scientific</td><td>12-565-94</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>10010031</td><td></td></tr><tr><td>PicoPump foot switch</td><td>World Precision Instruments</td><td>3260</td><td></td></tr><tr><td>Pipette tips, non-filtered, 1000 uL</td><td>Fisher Scientific</td><td>21-402-47</td><td></td></tr><tr><td>Pipette tips, non-filtered, 20 uL</td><td>Fisher Scientific</td><td>21-402-41</td><td></td></tr><tr><td>Pipette tips, non-filtered, 200 uL</td><td>Fisher Scientific</td><td>21-236-54</td><td></td></tr><tr><td>Pneumatic PicoPump system</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>or a similar picopump system</td></tr><tr><td>Primocin 50 mg/mL, 10x1 ml vial</td><td>InvivoGen</td><td>ant-pm-1</td><td></td></tr><tr><td>Steel base plate</td><td>World Precision Instruments</td><td>5052</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss stemi 350</td><td></td><td>Or a similar microscope</td></tr><tr><td>ThermoSafe PolarPack Foam Bricks</td><td>Sonoco</td><td>03-531-53</td><td></td></tr><tr><td>Triton X-100</td><td>Millipore Sigma</td><td>T8787</td><td></td></tr><tr><td>Wall air supply</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris Bioscience</td><td>1254</td><td></td></tr><tr><td>ZO-1 Polyclonal Antibody</td><td>Fisher Scientific</td><td>61-7300</td><td></td></tr></tbody></table>

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.

We established an enteroid cell line from donated ileum fetal tissue following modified protocols provided by our collaborators at the University of Michigan Translational Tissue Modeling Laboratory12. The enteroid cell line tested negative for Mycoplasma infection. The enteroids stained positive for villin, CDX2, lysozyme, mucin, claudin, occludin, and zonula-occluden 1 (Figure 5), confirming their small intestine epithelial origin. The enteroids were microinjected with 4 kDa dextran-FITC at 5 mg/mL in PBS (Figure 6). The test exposures were LPS at different concentrations, 0.1 mg/mL and 0.5 mg/mL, mixed with dextran-FITC for apical exposure. As a positive control, we used 2 mM EGTA and added it to the culture media at 4 h post microinjection of dextran. EGTA is a calcium chelator that increases the permeability of tight junctions. The negative controls were microinjected with dextran-FITC in PBS alone. For basolateral exposure, the replacement media for serial culture media collection had the same concentration of the exposure (i.e., 2 mM EGTA). The results show a clear increase in dextran concentration in culture media after the addition of EGTA in comparison to the negative control, PBS. Apical exposure of LPS induced higher permeability of dextran starting approximately 8 h post exposure in a concentration-dependent manner (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64108/64108fig01.jpg" /> 
Figure 1: Microinjector setup. The setup includes a stereo microscope, micromanipulator mounted on a magnetic stand on a heavy steel base plate, a micropipette holder connected to a syringe with a three-way stopcock, and a pneumatic pump. The wall air supply provides the air pressure, which is regulated by the pump to generate a consistent pump volume. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64108/64108fig02.jpg" /> 
Figure 2: Horizontal micropipette holder. This plastic platform is designed to hold the micropipette after pulling of the glass capillary tube for cutting the tip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64108/64108fig03.jpg" /> 
Figure 3: Micropipette positioning. An angle of 35&#176;-45&#176; is ideal for injecting enteroids located in the middle of the Petri dish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64108/64108fig04.jpg" /> 
Figure 4: Dextran standard curve. The curve was constructed with fluorescent absorbance of 10 serial dilutions of dextran in duplicates. A linear regression line was fitted to generate a regression equation. The error bars are SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64108/64108fig05.jpg" /> 
Figure 5: Fluorescent biomarkers of enteroids. DAPI for nucleus stain is blue. The following biomarkers are shown: (A) villin, (B) CDX2, (C) lysozyme, (D) mucin, (E) claudin, (F) occluding, and (G) zonula-occluden 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64108/64108fig06.jpg" /> 
Figure 6: Enteroids at different stages. (A) An enteroid at 7-10 days old is small and with a thick wall. (B) An enteroid that is ready for microinjection with a large size, a lumen, and a think wall, and (C) fluorescent dextran-FITC inside an enteroid 2 days after microinjection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64108/64108fig07.jpg" /> 
Figure 7: Permeability of dextran-FITC post microinjection. (A) Measured dextran levels in the media were higher after the addition of EGTA compared to PBS. (B) Microinjected 0.5 mg/mL LPS induced greater leakage of dextran from the enteroid lumen into the media than microinjected 0.1 mg/mL LPS did beginning at 8 h post-injection. *p-value &#60; 0.05, error bars are SEM, n = 3, a Student&#39;s t-test was used to calculate significance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64108/64108fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

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 ...

testing-epithelial-permeability-in-fetal-tissue-derived-enteroids

Nanyang Technological University

Research

JoVE Journal

Medicine

2.0K Views.  University of South Florida. Het belang van dit protocol is om de bruto waarschijnlijkheid van enteroïden te testen op het in vitro model van voortijdig epitheel. Het belangrijkste voordeel van deze techniek is het meten van de permeabiliteit van een ingesloten lumen dat de darmluminale omgeving nabootst. Dit protocol kan worden gebruikt om de factoren te bestuderen die lekkende darmziekte induceren of verzwakken.Het kan ook worden toegepast op endotheel- of vasculaire systemen om aandoeningen te bestuderen die v...

Video: Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Bioengineering

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

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

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

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

Developmental Biology

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

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

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

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

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

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

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

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

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

Immunology and Infection

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

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

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

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

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

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

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

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

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 ...

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

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