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>

<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. 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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 steps in this protocol. Tissue must be collected immediately at the time of operative resection and processed for crypt isolation as soon as the tissue arrives in the lab. We selected tissue from patients less than 3 months old for this model. Delayed processing can occur within 24 h after whole tissue collection if needed. Additionally, the quality of the human tissue sample greatly affects the yield of crypt isolation. Healthy small intestine without underlying pathology and from younger patients (&#60;2 months of age) have been found to yield the best results. Additionally, the collected tissue should be chosen from the healthiest area, typically at the distal ends of the resected specimen. Using a larger piece of intestine does not improve crypt isolation with the described solution volumes in the protocol above. Intestinal segments approximately 1-2 cm in length, weighing 0.75-2.5 g are optimal. The induction of experimental NEC via LPS exposure was modeled from well-established cell culture and animal models. The established work of Hackam et al. showed the critical role of TLR4 (LPS receptor) in NEC development9,13,14,15. LPS activation of TLR4 stimulates proinflammatory cytokines, a reduction in barrier integrity, and activation of subepithelial leukocytes that characterize the signaling events involved in human NEC. TLR4 has been implicated as a key molecule in promoting inflammation7 and animals that lack functional TLR4 are have demonstrated protection from the development of NEC15. Circulating levels of LPS are elevated in patients with NEC and elevated in stool and plasma of animal models of NEC. LPS induces intestinal inflammation in animals that resembles human NEC, which highlights the significance of inflammation in this pathway. Mirroring the established cell culture and animal models of NEC, we believe that induction of experimental NEC via LPS administration in enteroid culture is a useful ex vivo human neonatal model for the study of NEC. In line with previous reports in other established models, our experimental NEC enteroids demonstrated increased expression of TLR4 compared to controls.
The protocol for induction of experimental NEC via LPS exposure has undergone several important modifications and improvements. Initially, enteroids were grown for 5 days, then inoculated with LPS for 24 h and subsequently collected. The protocol now recommends LPS inoculation on day 0 with continued exposure in every culture media change until collection. Additionally, the dosage of LPS was optimized. After performing a dose curve and trialing several different LPS concentrations, 5 mg/mL was selected. Experimentation with LPS inoculation directly into the basement membrane matrix/enteroid mixture at the time of plating was also trialed; however, this was cumbersome and did not improve results.
There are limitations that should be addressed as the utilization of the human enteroid model evolves. Since the discovery in 2007, several different protocols have been used to establish and culture enteroids. The numerous growth factors required for enteroid maintenance and differentiation lack standardization, which may affect reproducibility. Additionally, enteroid culture lacks mechanical forces that affect the human intestinal epithelium in physiologic conditions. Pressure from luminal flow and peristalsis may influence gene and protein expression, which is not accounted for in this model. This model also lacks nerves, immune cells, a microbiome, vasculature and mesenchyme that are present in human intestinal epithelium.
Exposure to LPS in this human intestinal enteroid model causes an inflammatory response leading to histologic, genetic, and protein expression alterations similar to those found in human NEC. Although there are several current animal models of NEC, response to intestinal injury and therapeutic interventions varies widely between species. Since it is ethically challenging to study disease pathophysiology or to test novel therapeutic agents directly in humans, especially infants, it is exceedingly important to continue to cultivate this novel ex vivo model of NEC using human tissue. Human enteroids are amenable to genetic modification and may be fundamental in the development of therapies for many human intestinal diseases16. Future directions should aim to culture human enteroids on a permeable scaffold in a perfusion chamber with apical and basolateral flow to in order to reproduce in vivo crypt-villus architecture as well as physiologic forces such as peristalsis and luminal flow.

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 time.
The growing enteroids will also exhibit polarity, containing a centralized lumen, an apical border and a basolateral domain (Figure 2). Enteroids also show structural integrity, represented by a robust actin cytoskeleton (Figure 3). After several days in culture, the LPS treated enteroids experience more apoptosis and will have a lower yield than the control group (Figure 4). As seen in human NEC and murine models of NEC9, an increased expression of Toll-like receptor 4 (TLR4) was found in LPS treated enteroids compared to controls (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59194/59194fig1.jpg" /> 
Figure 1:&#160;Crypt culture and human enteroid formation from whole tissue. (a) Day 0 crypt culture with round, flat appearance. (b) Day 4 enteroids with spheroid formation. (c) Day 7 enteroids with budding (black arrow). Scale bars = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59194/59194fig1large.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/59194/59194fig2.jpg" /> 
Figure 2: Histological appearance of an enteroid. Haemotoxylin and Eosin staining. The enteroid displays a centralized lumen and exhibits polarity, with both an apical and basolateral domain. The haemotoxylin (purple) represents the nucleus, whereas the eosin (pink) represents cytosolic components. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59194/59194fig2large.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/59194/59194fig3.jpg" /> 
Figure 3: Enteroid actin cytoskeleton. Immunoflorescence micrograph of an enteroids. The enteroid displays structural integrity as demonstrated by the prominent actin cystoskeleton (magenta). Nuclei stained with DAPI (blue), Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59194/59194fig3large.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/59194/59194fig4.jpg" /> 
Figure 4:&#160;Human enteroid culture yield is decreased with LPS treatment. Day 5 enteroids in culture, grown from the same whole tissue sample. (a) Enteroids treated with control culture media. Display robust growth. (b) Enteroids treated with LPS in culture media. Only a few surviving enteroids. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59194/59194fig4large.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/59194/59194fig5.jpg" /> 
Figure 5:&#160;Increased TLR4 expression in experimental NEC enteroids. (a) qRT-PCR showed increased TLR4 mRNA expression in enteroids exposed to LPS (p = 0.03). (b) Western blot analysis showing increased TLR4 expression in experimental human enteroid NEC (p = 0.02). Representative western blot depicted. Values are means &#177; SEM of 3 samples per group. *p &#60; 0.05 by Student&#39;s t test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59194/59194fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

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JoVE Journal

Immunology and Infection

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 ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

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

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

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens