Name: a novel human epithelial enteroid model of necrotizing enterocolitis
Uploaded: 2019-04-10T13:00:00
Description: Watch this Scientific Journal Video about A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis at JoVE.com

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
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	<li>Prepare Culture Media st...

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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.

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			<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 ...

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