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 st...

<ol>
	<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>Zachos, N. 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. 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=Enterocyte+TLR4+mediates+phagocytosis+and+translocation+of+bacteria+across+the+intestinal+barrier.">Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier.</a> The Journal of Immunology. 176 (5), 3070-3079 (2006).</li><li>Sodhi, C. P., 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+epithelial+Toll-like+receptor+4+regulates+goblet+cell+development+and+is+required+for+necrotizing+enterocolitis+in+mice.">Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.</a> Gastroenterology. 143 (3), 708-718 (2012).</li><li>Koo, B. 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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9 (1), 81-83 (2011).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:887px;" width="887">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">4% Paraformaldehyde</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">AAJ19943K2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">A-83</td>
			<td style="width:139px;">R&#38;D Tocris</td>
			<td style="width:167px;">2939/10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Amphotericin B</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">15290026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">B-27 supplement minus Vitamin A</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Basement Membrane Matrix (Matrigel)</td>
			<td style="width:139px;">Corning</td>
			<td style="width:167px;">CB-40230C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">DMEM/F-12</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">MT-16-405-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">11-965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Dulbecco&#8217;s Phosphate-Buffered Saline (DPBS)</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Epidermal Growth Factor (EGF)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">E9644-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">EDS-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gemini Bio-Pro</td>
			<td style="width:167px;">100-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Gentamicin</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">G5013-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">GlutaMAX (L-glutamine)</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Insulin</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">I9278-5mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">[leu] 15-gastrin 1</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">G9145-.1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Lipopolysaccharide (LPS)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">L2630-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">N-2 supplement</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">N-Acetylcysteine</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">A9165-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Nicotinamide</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">N0636-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Noggin</td>
			<td style="width:139px;">R&#38;D Systems INC</td>
			<td style="width:167px;">6057-NG/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Penicillin-Streptomycin</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:167px;">15140-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Phosphate Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>P5368-5X10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">RPMI 1640 Medium</td>
			<td style="width:139px;">Invitrogen</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">R-Spondin</td>
			<td style="width:139px;">PEPROTECH INC</td>
			<td style="width:167px;">120-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">SB202190</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">S7067-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Processing Gel (Histogel)</td>
			<td style="width:139px;">ThermoFisher</td>
			<td>22-110-678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Wnt3a</td>
			<td style="width:139px;">R&#38;D Systems INC</td>
			<td style="width:167px;">5036-WN-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Y-27632</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:167px;">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...

Watch this Scientific Journal Video about A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis at JoVE.com

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 ...

Enteroids are three dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiological interactions, cell signaling, and host pathogen defense. Our protocol describes how to culture human enteroids after isolating intestinal stem cells from patients undergoing bowel resection.Co-administration of lipopolysaccharide in our culture media causes an inflammatory response in the enteroids leading to histologic, genetic, and protein expression alterations similar to those seen in human necrotizing enterocolitis. Given that the study of necrotizing enterocolitis and potential therapeutic agents is ethically challenging in human subjects, especially children, it is highly desirable to utilize this novel, biologically relevant enteroid model of necrotizing enterocolitis using human neonatal tissue. The main advantage of this technique is that enteroids are able to recapitulate the multiple cells types of human intestinal epithelium and are able to overcome the recognized limitations of animal models and cell based systems.Helping to demonstrate the procedure will be Christie Buonpane, a surgical resident, and Carrie Yuan, a laboratory technician from our laboratory. At the time of collection in the operative suite, place the human small intestinal tissue sample in cold DPBS. Wash the specimen in the cold DPBS until it is clear of blood and stool.Store the specimen in RPMI 1640 medium at four degrees Celsius until ready for crypt isolation. When ready to proceed, check again that the specimen is clear of stool and blood. Using delicate dissecting scissors remove any excess fat or surgical clips and staples.Weight the specimen and aim for a piece that is approximately 0.75 to 2.5 grams. Next, cut the tissue into 0.5 centimeter pieces and place them in a tube containing 30 milliliters of chelating buffer number one. Shake at a low speed for 15 minutes at four degrees Celsius.Then, filter the tissue through a 100 micrometer cell strainer and discard the flow through. Add the filtered tissue to a tube containing 30 milliliters of chelating buffer number two. Shake at a low speed for 15 minutes at four degrees Celsius.Filter the tissue through a 100 micrometer filter and discard the flow through. After this, thaw 500 microliters of basement membrane matrix on ice for later use. Add some tissue to 10 milliliters of cold DMEM in a 50 milliliter conical tube and shake vigorously by hand for 10 seconds.Filter this suspension through a 100 micrometer cell strainer and collect the flow through. Keep this tube on ice. Add some tissue to another 10 milliliters of cold DMEM in a separate 50 milliliter conical tube and shake vigorously by hand for 10 seconds.Filter this suspension through a 100 micrometer cell strainer and collect the flow through. Repeat the process two additional times until there are four conical tubes containing flow through labeled one through four. Then, filter the solution in tube number one through a 100 micrometer cell strainer and transfer the flow through into a 15 milliliter conical tube also labeled number one.Repeat this process for tubes two through four. Centrifuge the 15 milliliter tubes at 200 times G and at four degrees Celsius for 15 minutes. In the laminar flow hood, remove the supernatant from each tube and discard it.Avoid disrupting the cloud of tissue immediately above the pellet, even if that means leaving some supernatant behind. In each tube, pipette up and down slowly to mix the pellet with the leftover supernatant. Transfer the mixture from each tube into a single two milliliter conical tube and centrifuge at 200 times G and at four degrees Celsius for 20 minutes.After this, remove the supernatant and resuspend the pellet in 500 microliters of pre-thawed basement membrane matrix. Use a chilled pipette tip to apply 50 microliters of this suspension to the center of a well in a 24 well plate. This sample should appear dome shaped.Repeat this application process nine times to fill 10 total wells. Transfer the 24 well plate into a 5%carbon dioxide incubator at 37 degrees Celsius for 30 minutes to allow polymerization. Then, add 500 microliters of human minigut media complete to each well.Continue incubating under the same conditions, making sure to replace this media every two days. First, add 10 microliters of LPS at a concentration of five milligrams per milliliter to the 500 microliters of human minigut media complete in each well of the plate on day zero. Continue incubating at 37 degrees Celsius with 5%carbon dioxide and replace the supplemented media every two days until collection.To prepare for paraffin embedding, first gently remove the media, add one milliliter of PBS, and gently pipette up and down to dissolve the basement membrane matrix while being careful not to lyse the enteroids. Centrifuge at a speed less than 300 times G for five minutes to pellet and then remove the PBS. Add 4%paraformaldehyde and set the tube aside for one hour to fix at room temperature.Centrifuge at a speed less than 300 times G for five minutes to pellet and then remove the paraformaldehyde. Wash gently with one milliliter of PBS and centrifuge at a speed less than 300 times G for five minutes. Then remove the PBS.Repeat this wash process with PBS once. Place the desired amount of the tissue processing gel into a conical tube and warm it in a dry bath incubator at 65 degrees Celsius for three to 10 minutes until it is liquid. After this, add the melted tissue processing gel to the pellet and mix gently.Place a small cloning ring into a coverslip. Pipette the tissue processing gel and enteroid mixture into the cloning ring that is mounted onto a coverslip. Allow the tissue processing gel and the enteroid mixture to solidify at four degrees Celsius for one hour.Then, submerge the cloning ring with the solidified mixture into 70%ethanol to prepare for paraffin embedding. Immediately after plating, the freshly isolating intestinal crypts appear as elongated rods. Within hours the enteroids will take on a round appearance.Over the next several days the enteroids will start forming spheres. Budding should occur between five to 10 days, which is when enteroid collection should occur. The growing enteroids also exhibit polarity, containing a centralized lumen, an apical border, and a basilateral domain.Enteroids also show structural integrity represented by a robust actin cytoskeleton. After several days in culture, the LBS treated enteroids experience more apoptosis and have a lower yield than the control group. As seen in human NEC in murine models of NEC, an increased expression of toll like receptor four is found in LPS treated enteroids compared to controls.The enteroids can also be collected for RNA and protein extraction using well established qRT-PCR and western blotting techniques gene expression and protein isolation can be performed.

a-novel-human-epithelial-enteroid-model-of-necrotizing-enterocolitis

Research

JoVE Journal

Immunology and Infection

7.5K visualizações.  Northwestern University. Os enteróides são organoides tridimensionais derivados de células epiteliais intestinais. São ideais para o estudo de interações fisiológicas complexas, sinalização celular e defesa de patógenos hospedeiros. Nosso protocolo descreve como cultivar enteróides humanos depois de isolar células-tronco intestinais de pacientes submetidos à ressecção intestinal.A coadministração de lipopolisacarídeo em nossa mídia cultural causa uma resposta inflamatória nos enteróides le...