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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+birth+and+sustained+inflammation:+Consequences+for+the+neonate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+Model+systems+for+human+biology+and+medicine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canonical+Wnt+signals+are+essential+for+homeostasis+of+the+intestinal+epithelium.">Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.</a> Genes &amp; Development. 17 (14), 1709-1713 (2003).</li><li>Barker, 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">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/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 ...

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

Research

JoVE Journal

Medicine

2.0K Views.  University of South Florida. 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. 

Video: Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

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 ...