This work was supported by the National Institute of Health Institute of Diabetes and Digestive and Kidney Disease Grant (K08DK106450) and the Jay Grosfeld Award from the American Pediatric Surgical Association to C.J.H.

Institutional review board approval was obtained (IRB #2013-15152) for collection of tissue samples from patients undergoing bowel resection at Ann and Robert H. Lurie Children&#39;s Hospital of Chicago, Chicago, IL. All protocols were performed in compliance with institutional and national guidelines and regulations for human welfare. Written informed parental consent was required and obtained prior to sample collection in all cases.
1. Reagent Preparation
<ol>
	<li>Prepare Culture Media stock solution for whole tissue collection: 1 L of Dulbecco&#39;s Modified Eagle Medium (DMEM), 110 mL of Fetal Bovine Serum (FBS), 11 mL of Penicillin-Streptomycin (final concentration 1%) and 1.1 mL of filter-sterilized insulin (final concentration 0.1%.) Store stock solution at 4 &#176;C.</li>
	<li>Prepare Chelating Buffer #1: 30 mL of Culture Media (as described in 1.1), 600 &#181;L of 0.5 M Ethylenediaminetetraacetic acid (EDTA) (final concentration 10 mM), 300 &#181;L of Gentamicin (final concentration 1%) and 60 &#181;L of Amphotericin B (final concentration 0.2%). Store stock solution at 4 &#176;C.</li>
	<li>Prepare Chelating Buffer #2: 30 mL of Culture Media (as described in 1.1), 300 &#181;L of 0.5 M EDTA (final concentration 5mM), 300 &#181;L of Gentamicin (final concentration 1%) and 60 &#181;L of Amphotericin B (final concentration 0.2%). Store stock solution at 4 &#176;C.</li>
	<li>Prepare Human Minigut Media: 41.4 mL of DMEM/F-12, 5 mL of FBS (final 10%), 500 &#181;L of Penicillin-Streptomycin (final concentration 1%), 500 &#181;L of L-glutamine (final concentration 1%), 500 &#181;L of Gentamicin (final concentration 1%), 100 &#181;L of Amphotericin B (final concentration 0.2%), 500 &#181;L of 1 M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (final concentration 10 mM), 500 &#181;L of 100x N-2 supplement, and 1 mL of 50x B-27 supplement minus Vitamin A for a total volume of 50 mL. Store stock solution at -20 &#176;C.</li>
	<li>Prepare Human Minigut Media Complete: 10 mL of Human Minigut Media (prepared in 1.4), 10 &#181;L of 100 &#181;g/mL Wnt3a (final concentration 100 ng/mL), 10 &#181;L of 100 &#181;g/mL Noggin (final concentration 100 ng/mL), 10 &#181;L of 1 mg/mL R-Spondin (final concentration 1 &#181;g/mL), 10 &#181;L of 500 &#181;g/mL Epidermal Growth Factor (EGF) (final concentration 50 ng/mL), 10 &#181;L of 1 M N-Acetylcysteine (final concentration 1 mM), 10 &#181;L of 10 mM Y-27632 (final concentration 10 &#181;M), 10 &#181;L of 500 &#181;M A-83 (final concentration 500 nM), 10 &#181;L of 10 mM SB202190 (final concentration 10 &#181;M), 100 &#181;L of 1 M Nicotinamide (final concentration 10 mM) and 1 &#181;L of 100 &#181;M [leu] 15-gastrin 1 (final concentration 10 nM). Total volume 10 mL. Store stock solution at 4 &#176;C. 
	NOTE: Solutions must be used within 48 h.</li>
</ol>
2. Crypt Isolation and Plating from Whole Tissue
<ol>
	<li>At the time of collection in the operative suite, place the human small intestinal tissue sample in cold Dulbecco&#39;s Phosphate Buffered Saline (DPBS). Wash the specimen in cold DPBS until clear of stool and blood. Store specimen at 4 &#176;C in RPMI 1640 Medium until ready for crypt isolation. 
	NOTE: Tissue cannot be stored more than 24 h.
	<ol>
		<li>Make sure that the specimen is clear of stool and blood. Using delicate dissecting scissors, remove any excess fat or surgical clips/staples etc. Weigh the specimen. 
		NOTE: Aim for a piece approximately 0.75-2.5 g.</li>
	</ol>
	</li>
	<li>Cut the specimen into 0.5 cm pieces and place in 30 mL of Chelating Buffer #1 (as prepared in step 1.2).
	<ol>
		<li>Shake at low speed for 15 min at 4 &#176;C.</li>
		<li>Filter tissue through 100 &#181;m cell strainer and discard flow through.</li>
	</ol>
	</li>
	<li>Add the tissue to 30 mL of Chelating Buffer #2 (as prepared in step 1.3).
	<ol>
		<li>Shake at low speed for 15 min at 4 &#176;C.</li>
		<li>Filter tissue through a 100 &#181;m cell strainer and discard flow through.</li>
	</ol>
	</li>
	<li>Thaw 500 mL of basement membrane matrix on ice for use in step 2.8.</li>
	<li>Add tissue to 10 mL of cold DMEM in a 50 mL conical tube and shake vigorously by hand for 10 s.
	<ol>
		<li>Filter through a 100 &#181;m cell strainer and collect flow through (Label #1). Keep tube #1 on ice.</li>
		<li>Add tissue to another 10 mL of cold DMEM in a separate 50 mL conical tube and shake vigorously by hand for 10 s.</li>
		<li>Filter through a 100 &#181;m cell strainer and collect flow through (label #2).</li>
		<li>Repeat two additional times until there are four conical tubes with flow through (labeled tubes #1-4).</li>
	</ol>
	</li>
	<li>Filter tube #1 solution through a 100 &#181;m cell strainer and transfer flow through into 15 mL conical tube (Label #1). Repeat for #2-4.
	<ol>
		<li>Centrifuge 15 mL tubes #1-4 at 200 x g for 15 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>In the laminar flow hood, remove the supernatant from tubes #1-4 and discard. Avoid disrupting the cloud of tissue immediately above the pellet, even if that means leaving some supernatant behind.
	<ol>
		<li>By pipetting slowly, mix together the pellet with the leftover supernatant in tubes #1-4.</li>
		<li>Transfer the mixture from tubes #1-4 into one single 2 mL conical tube.</li>
		<li>Centrifuge the conical tube at 200 x g for 20 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Remove the supernatant and re-suspend the pellet in 500 &#181;L of basement membrane matrix. 
	NOTE: Keep the basement membrane matrix on ice at all times and work quickly for the next steps. This product polymerizes very quickly at room temperature.
	<ol>
		<li>Apply 50 &#181;L of specimen/basement membrane matrix suspension to the center of a well in a 24-well plate. This should appear dome-shaped. 
		NOTE: Use of chilled pipette tips aids in smoother transfer of the suspension, minimizing polymerization.</li>
		<li>Repeat 9 times to fill 10 total wells.</li>
	</ol>
	</li>
	<li>Place the 24-well plate in 37 &#176;C, 5% CO2 incubator for 30 min to allow polymerization.</li>
	<li>Add 500 &#181;L of Human Minigut Media Complete (as prepared in step 1.5) to each well. Replace every 2 days.</li>
	<li>Collect enteroids after 5-10 days, when budding is visualized. See Steps 4 and 5 for collection instructions.</li>
</ol>
3. Induction of Experimental NEC
<ol>
	<li>Add 10 &#181;L of 5 mg/mL of lipopolysaccharide (LPS) to 500 &#181;L of Human Minigut Media Complete (as prepared in step 1.5) in each well on Day 0. Replace every 2 days until collection.</li>
</ol>
4. Preparation for Paraffin Embedding
<ol>
	<li>Gently remove media.
	<ol>
		<li>Add 1 mL of PBS and gently pipette up and down to dissolve the basement membrane matrix with care not to lyse the enteroids.</li>
		<li>Centrifuge PBS/enteroid mixture at &#60;300 x g for 5 min to pellet and remove PBS.</li>
	</ol>
	</li>
	<li>Add 4% paraformaldehyde to fix at room temperature for 1 h.</li>
	<li>Centrifuge at &#60;300 x g for 5 min to pellet, and then remove PBS.
	<ol>
		<li>Wash gently with 1 mL of PBS and centrifuge at &#60;300 x g for 5 min to pellet, and then remove PBS.</li>
		<li>Repeat step 4.3.1.</li>
	</ol>
	</li>
	<li>Melt a sufficient volume of the tissue processing gel (around 300 &#181;L) by placing the desired amount in a conical tube and warming it in a dry bath incubator at 65 &#176;C for 3-10 min until liquid.
	<ol>
		<li>Add the tissue processing gel to the pellet and mix gently.</li>
		<li>On a coverslip, place a small cloning ring.</li>
		<li>Pipette the tissue processing gel and enteroid mixture into a cloning ring mounted onto a coverslip.</li>
	</ol>
	</li>
	<li>Allow the tissue processing gel and enteroid mixture to solidify at 4 &#176;C for 1 h.
	<ol>
		<li>Submerge the cloning ring with solidified mixture into 70% ethanol in preparation for paraffin embedding.</li>
	</ol>
	</li>
</ol>
5. Enteroid Collection for RNA and Protein Extraction
<ol>
	<li>Gently remove Human Minigut Media Complete from each well.</li>
	<li>Add 1 mL PBS and gently pipette up and down to dissolve the basement membrane matrix. Be careful not to dissociate the enteroids.
	<ol>
		<li>Place PBS/enteroid mixture into a sterile 2 mL conical tube.</li>
		<li>Centrifuge at &#60;300 x g for 5 min to pellet.</li>
		<li>Gently remove PBS, taking care not to remove the enteroids.</li>
	</ol>
	</li>
	<li>Repeat step series 5.2 two more times.</li>
	<li>Freeze in -80 &#176;C until ready for protein and/or RNA extraction.</li>
	<li>Perform gene expression and protein isolation of the enteroids using well-established qRT-PCR and Western Blotting techniques.</li>
</ol>

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C., 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+Enteroids/Colonoids+and+Intestinal+Organoids+Functionally+Recapitulate+Normal+Intestinal+Physiology+and+Pathophysiology.">Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.</a> The Journal of Biological Chemistry. 291 (8), 3759-3766 (2016).</li><li>Foulke-Abel, J., 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+enteroids+as+an+ex-vivo+model+of+host-pathogen+interactions+in+the+gastrointestinal+tract.">Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract.</a> Experimental Biology and Medicine (Maywood). 239 (9), 1124-1134 (2014).</li><li>Hidalgo, I. J., Raub, T. J., Borchardt, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+human+colon+carcinoma+cell+line+(Caco-2)+as+a+model+system+for+intestinal+epithelial+permeability.">Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability.</a> Gastroenterology. 96 (3), 736-749 (1989).</li><li>Hilgers, A. R., Conradi, R. A., Burton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cell+monolayers+as+a+model+for+drug+transport+across+the+intestinal+mucosa.">Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa.</a> Pharmaceutical Research. 7 (9), 902-910 (1990).</li><li>In, J. G., 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+mini-guts:+new+insights+into+intestinal+physiology+and+host-pathogen+interactions.">Human mini-guts: new insights into intestinal physiology and host-pathogen interactions.</a> Nature Reviews Gastroenterology &amp; Hepatology. 13 (11), 633-642 (2016).</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> Cell and Molecular Gastroenterology and Hepatology. 5 (4), 549-568 (2018).</li><li>Moore, S. 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=Intestinal+barrier+dysfunction+in+human+necrotizing+enterocolitis.">Intestinal barrier dysfunction in human necrotizing enterocolitis.</a> Journal of Pediatric Surgery. 51 (12), 1907-1913 (2016).</li><li>Neal, M. D., 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+critical+role+for+TLR4+induction+of+autophagy+in+the+regulation+of+enterocyte+migration+and+the+pathogenesis+of+necrotizing+enterocolitis.">A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis.</a> The Journal of Immunology. 190 (7), 3541-3551 (2013).</li><li>Lanik, W. E., 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=Breast+Milk+Enhances+Growth+of+Enteroids:+An+Ex+Vivo+Model+of+Cell+Proliferation.">Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation.</a> Journal of Visualized Experiments. (132), 56921 (2018).</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), 52483 (2015).</li><li>Uzarski, J. S., Xia, Y., Belmonte, J. C., Wertheim, 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=New+strategies+in+kidney+regeneration+and+tissue+engineering.">New strategies in kidney regeneration and tissue engineering.</a> Current Opinion in Nephrology and Hypertension. 23 (4), 399-405 (2014).</li><li>Leaphart, C. L., 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+critical+role+for+TLR4+in+the+pathogenesis+of+necrotizing+enterocolitis+by+modulating+intestinal+injury+and+repair.">A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair.</a> The Journal of Immunology. 179 (7), 4808-4820 (2007).</li><li>Neal, M. 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The authors have nothing to disclose.

This novel ex vivo human intestinal enteroid model serves as a useful method for the study of intestinal barrier dysfunction in necrotizing enterocolitis (NEC). The enteroid processing methods presented here were adapted from the previous work of Drs. Misty Good, Michael Helmrath and Jason Wertheim10,11,12.
Details surrounding the whole tissue collection and timing of crypt isolation are critical step...

Human enteroids are an ex vivo 3-dimensional culture system generated from stem cells isolated from intestinal crypts of human intestinal tissue samples. This ground-breaking model was pioneered by Hans Clevers et al. in 2007 following the discovery of Lgr5+ stem cells at the crypts of small intestine in mice1. Their work laid the foundation for establishing an ex vivo intestinal epithelial culture of multiple cell types that could be passaged without significant genetic or physiologic changes2. Since this discovery, enteroids have been used as a novel model to study normal digestive physiology, and the pathophysiology of intestinal diseases such as inflammatory bowel disease, host-pathogen interactions, and regenerative medicine2.
The use of enteroids as an ex vivo model for the study of intestinal pathophysiology has several advantages over alternative techniques. For the past several decades, animal models and immortalized intestinal cancer-derived cell lines have been used to study intestinal physiology3,4,5. Single-cell cultures do not represent the diversity of cell types present in normal intestinal epithelium, thereby lacking cell to cell cross-talk and segment-specificity in protein expression, signaling, and pathogen-induced disease6. Stem cells in enteroids differentiate into the major epithelial cell types such as enterocytes, Paneth cells, goblet cells, enteroendocrine cells and more3. They exhibit polarity, carry out epithelial transport functions, and allow for intestinal segment specificity6. Since enteroids can recapitulate the multiple cell types of human intestinal epithelium, they are able to overcome this recognized limitation of cancer cell-based systems. Over time, derivatives of cell lines are subcloned and evolve to exhibit greater diversity in protein expression and localization3. On the contrary, enteroids can be passaged without significant genetic or physiologic changes2. Although numerous animal models for NEC exist, response to injury and therapeutic interventions may be highly variable between species. As a result of these limitations, therapeutics derived from animal models fail 90% of the time when tested in human trials due to differences in toxicity or efficacy3. Enteroids serve as promising pre-clinical models that can overcome these deficiencies, leading to a better understanding of complex intestinal pathophysiology and therefore, more successful and cost-effective therapeutic innovations. There is also recent evidence that the age of the tissue that an enteroid is generated from remains biologically important7. This is an especially important detail for our model since enteroids are generated from neonatal tissue, thereby maintaining physiologic relevance to patients with NEC.
The utility of enteroids as models of human illnesses continues to expand, in hopes of finding cures to severe and pervasive conditions. Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates characterized by intestinal necrosis and frequently leads to perforation of the bowel wall, septicemia, and death8. Due to the complex and multifactorial pathophysiology of NEC, the exact mechanism of the disease has not yet been fully elucidated; however, increased intestinal permeability has been clearly implicated in the disease process8. Given that the study of NEC and potential therapeutic agents is ethically challenging in human subjects, especially children, it is highly desirable to utilize a biologically relevant enteroid model of NEC using human neonatal tissue. Thus far, enteroids have a limited role in the study of NEC. This protocol describes the use of enteroids derived from human intestinal tissue samples as a novel ex vivo model for the study of necrotizing enterocolitis.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>ThermoFisher</td><td>AAJ19943K2</td><td></td></tr><tr><td>A-83</td><td>R&amp;amp;D Tocris</td><td>2939/10</td><td></td></tr><tr><td>Amphotericin B</td><td>ThermoFisher</td><td>15290026</td><td></td></tr><tr><td>B-27 supplement minus Vitamin A</td><td>ThermoFisher</td><td>17504-044</td><td></td></tr><tr><td>Basement Membrane Matrix (Matrigel)</td><td>Corning</td><td>CB-40230C</td><td></td></tr><tr><td>DMEM/F-12</td><td>ThermoFisher</td><td>MT-16-405-CV</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>11-965-118</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline (DPBS)</td><td>ThermoFisher</td><td>14190-144</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Sigma</td><td>E9644-.2MG</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>EDS-500G</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gemini Bio-Pro</td><td>100-125</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G5013-1G</td><td></td></tr><tr><td>GlutaMAX (L-glutamine)</td><td>ThermoFisher</td><td>35050-061</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I9278-5mL</td><td></td></tr><tr><td>[leu] 15-gastrin 1</td><td>Sigma</td><td>G9145-.1MG</td><td></td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma</td><td>L2630-25MG</td><td></td></tr><tr><td>N-2 supplement</td><td>ThermoFisher</td><td>17502-048</td><td></td></tr><tr><td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)</td><td>ThermoFisher</td><td>15630-080</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma</td><td>A9165-5G</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636-100G</td><td></td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems INC</td><td>6057-NG/CF</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher</td><td>15140-148</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P5368-5X10PAK</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Invitrogen</td><td>11875093</td><td></td></tr><tr><td>R-Spondin</td><td>PEPROTECH INC</td><td>120-38</td><td></td></tr><tr><td>SB202190</td><td>Sigma</td><td>S7067-5MG</td><td></td></tr><tr><td>Tissue Processing Gel (Histogel)</td><td>ThermoFisher</td><td>22-110-678</td><td></td></tr><tr><td>Wnt3a</td><td>R&amp;amp;D Systems INC</td><td>5036-WN-010</td><td></td></tr><tr><td>Y-27632</td><td>Sigma</td><td>Y0503-1MG</td><td></td></tr></tbody></table>

a novel human epithelial enteroid model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Immediately after plating, the freshly isolated intestinal crypts appear as elongated rods. Within hours, the enteroid will take on a round appearance (Figure 1a). Over the next several days, the enteroids will start forming spheres as seen in Figure 1b. Budding should occur between 5-10 days (Figure 1c) and enteroid collection should occur at that t...

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A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

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7.6K Views.  Northwestern University. Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

Video: A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Medicine

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model

Source: Ares, G. J., et al. <a href="https://app.jove.com/v/59194">A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.</a> J. Vis. Exp.(2019).
This video demonstrates the development of necrotizing enterocolitis disease in a human-derived neonatal enteroid model induced by lipopolysaccharides treatment. This model helps to study intestinal pathophysiology.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

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Chapters in this video

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model