Name: testing epithelial permeability in fetal tissue-derived enteroids
Uploaded: 2022-06-16T14:20:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids at JoVE.com

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>
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M., Sundaram, N., Watson, C. L., Shroyer, N. F., Helmrath, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+epithelial+enteroids+and+colonoids+from+whole+tissue+and+biopsy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+human+epithelial+enteroid+model+of+necrotizing+enterocolitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+human+intestinal+enteroid/organoid+lines+from+preterm+infant+and+adult+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+analysis+reveals+hallmarks+of+human+intestine+development+and+maturation+in+vitro+and+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal-derived+enterospheres+provide+insights+on+intestinal+development+and+a+novel+model+to+study+necrotizing+enterocolitis+(NEC).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+understanding+necrotizing+enterocolitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+colonic+crypts+in+culture:+Segregation+of+immunochemical+markers+in+normal+versus+adenoma-derived.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+colonization+stimulates+a+complex+physiological+response+in+the+immature+human+intestinal+epithelium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cryogenic+preservation+of+human+biopsy+specimens+and+subsequent+organoid+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Human+iPS+Cells+Into+Colon+Organoids+in+Serum-Free+Cell+Culture+Conditions.">In Vitro Differentiation of Human iPS Cells Into Colon Organoids in Serum-Free Cell Culture Conditions.</a> Technology Networks. , (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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurement+of+epithelial+barrier+permeability+in+human+intestinal+organoids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Intestinal+Barrier+and+Current+Techniques+for+the+Assessment+of+Gut+Permeability.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lineage-specific+transcription+factor+CDX2+navigates+dynamic+chromatin+to+control+distinct+stages+of+intestine+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prenatal+gut+microbiome:+Are+we+colonized+with+bacteria+in+utero.">The prenatal gut microbiome: Are we colonized with bacteria in utero.</a> Pediatric Obesity. 12, 3-17 (2017).</li><li>Spence, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+gradient+and+instability+of+Wnt+induce+heterogeneous+growth+and+differentiation+of+human+intestinal+organoids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Paneth+cell:+The+curator+and+defender+of+the+immature+small+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+colonoid+monolayers+to+study+interactions+between+pathogens,+commensals,+and+host+intestinal+epithelium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell-derived+models:+Tools+for+studying+role+of+microbiota+in+intestinal+homeostasis+and+disease.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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&#160;</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&#160;</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&#39;s Phosphate-buffered Saline (DPBS, 1X)</td>
			<td>Fisher Scientific</td>
			<td>AAJ67802K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-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&#34; (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&#160; Technologies</td>
			<td>&#160;06010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from&#160;Escherichia coli&#160;O111:B4</td>
			<td>Millipore Sigma</td>
			<td>L4391-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>World Precision Instruments&#160;</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix, LDEV-free, 10 mL&#160;</td>
			<td>Corning</td>
			<td>354234</td>
			<td>&#160;protein concentration &#62; 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&#160;</td>
			<td>M3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>World Precision Instruments&#160;</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>&#160;71-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 32% aqueous solution</td>
			<td>ELECTRON&#160;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/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/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/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/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/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/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/files/ftp_upload/64108/64108fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids at JoVE.com

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 ...

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