Name: in vitro apical-out enteroid model of necrotizing enterocolitis
Uploaded: 2022-06-08T14:45:00
Description: Watch this Scientific Journal Video about In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis at JoVE.com

This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. HC is supported by grant P20GM134973 from the National Institutes of Health. KB is supported by a Children&#39;s Hospital Foundation (CHF) and Presbyterian Health Foundation (PHF) grant. We thank the Laboratory for Molecular Biology and Cytometry Research at OUHSC for the use of the Core Facility, which provided confocal imaging.

All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. Small intestine convenience samples from a preterm non-human primate (NHP, 90% gestation, olive baboon, Papio anubis) were obtained following euthanasia for a separate study (Protocol #101523-16-039-I).
1. Establishment of apical-out enteroid NEC-in-a-dish model
<ol>
	<li>Preparation of media and enteroid treatments<br /...

The recent development of enteroid models derived from intestinal epithelial crypts allows for a more physiologically relevant in vitro tissue in which to study necrotizing enterocolitis pathogenesis. Despite including all major differentiated cell types of the intestinal epithelium, 3D enteroids are still subject to several significant limitations. The conventional, basolateral-out enteroids are suspended in 3D ECM hydrogel domes, the composition and density of which may limit normal diffusion within the tissue...

Necrotizing enterocolitis (NEC), a severe inflammatory disease of the small intestine occurring in up to 10% of preterm infants, is commonly associated with high morbidity and mortality1,2. Mortality rates approaching 50% in very low birth weight (&#60;1500 g) infants, requiring surgical intervention, are not uncommon3. While the exact etiology of NEC is not currently understood, risk factors, such as formula feeding, are thought to compound with physiological anomalies, such as dysbiosis, an immature intestinal epithelium, and a dysfunctional intestinal barrier, in the development of the disease2,4. Despite significant effort, little progress in the prevention or treatment of NEC has occurred over the last decade5. A novel in vitro method to study NEC and associated intestinal epithelial barrier dysfunction is required to advance understanding of the pathogenesis of the disease, as findings from animal models have, thus far, translated poorly to the bedside6.
A number of in vitro models have been utilized to investigate the mechanisms at play during NEC. The human intestinal epithelial cell line, Caco-2, is among the most commonly utilized in vitro models of NEC7,8. Caco-2 cells emulate brush border morphological features of the small intestine, but, as a cell line, do not differentiate into the wide variety of in vivo cell types, including mucus-producing goblet cells, required for a highly translatable model. HT-29-MTX, human colon adenocarcinoma cells, include a mixed enterocyte and goblet cell phenotype, but still lack crypt-based cell types of the intestinal epithelium9. IEC-6 and IEC-18 are non-transformed cell lines with an immature ileal crypt-like morphology but are not derived from human tissue, limiting their translational capacity. FHs 74-Int and H4 intestinal cell lines are derived from human fetal tissue and do not form tight junctions or polarized monolayers10,11, and thus are immature compared with even the most premature infants susceptible to NEC. Typically, NEC in vitro models utilize lipopolysaccharide (LPS) treatments to induce toll-like receptor 4 (TLR4), a major receptor initiating intestinal inflammation in NEC12. Damage mediated through reactive oxygen species (ROS) treatment, typically via hydrogen peroxide, is often used to induce NEC-like oxidative damage and apoptosis13,14. As the main driver of intestinal inflammation, tumor necrosis factor-alpha (TNF-&#945;), a downstream component of the inflammatory TLR4 signaling, is also commonly utilized in these in vitro models to mimic in vivo pathogenesis15.
Organoids, generated from inducible pluripotent stem cells (iPSCs), have grown in popularity as an in vitro model of the intestine due to their ability to recapitulate the complex in vivo architecture and cell-type composition of the tissue from which they are derived16,17. A related in vitro system, enteroids, are organoids derived from resected intestinal crypts that are more easily established and maintained than iPSC-derived organoids. Enteroids are typically grown in a three-dimensional (3D) extracellular matrix (ECM) with experimental access limited to the basolateral cell surface. Methods, such as microinjection18,19, have been developed to overcome this barrier to the apical surface, but the buildup of sloughed cellular debris and mucus within the lumen renders microinjection both technically difficult and inconsistent. As custom robotic microinjection platforms are not widely accessible20, lab-to-lab variability in technical ability and general technique become significant variables to overcome with microinjection protocols. Two-dimensional (2D) monolayers derived from dissociated 3D enteroids, still comprising all major cell types of the intestinal epithelium, allow access to the apical surface but have traditionally been difficult to maintain without a feeder layer of mesenchymal myofibroblasts21. While cell culture permeable supports can be used to access both the apical and basolateral sides of enteroid monolayers without the use of underlying myofibroblasts, these inserts require excision and mounting of the membrane before use with modalities such as confocal microscopy, resulting in a more technically demanding and difficult process when using traditional microscopy methods22. NEC has been modeled in vitro using traditional 3D enteroids23,24,25 and permeable supports26,27, and intestinal inflammation has recently been replicated with gut-on-a-chip models28,29. While gut-on-a-chip models incorporating microfluidics are, by far, the most advanced and translatable models, this technology is expensive, complex, and inaccessible to most investigators30.
Recent advances in apical-out enteroid techniques have allowed easier access to the apical surface of 3D enteroids without risking damage to the structural integrity of the in vitro epithelium31,32,33. Apical-out enteroids share the cell type composition and barrier function of in vivo intestinal epithelium, but, unlike typical 3D enteroids, the apical surfaces of these cells face the culture medium, allowing for more physiologically relevant studies on nutrient absorption, microbial infection, and luminal secretion31. An additional advantage of apical-out enteroids is the&#160;ability to homogenously distribute experimental agents to enteroids. Varying treatment volumes based on enteroid size is not required, as it is with microinjection, and the ability to maintain these enteroids in suspension culture negates any ECM interference on experimental agent diffusion32.
Necrotizing enterocolitis is a multifactorial disease involving multiple intestinal epithelial cell types and a variety of environmental and pathophysiological factors34. The varied cell composition of intestinal enteroids is a clear improvement over monocultures in modeling a complex disease such as NEC. Interestingly, while a single inflammatory exposure is often sufficient to induce damage in in vitro monocultures, enteroids, as with mouse models23, appear to require a minimum of two inflammatory components to induce NEC-like damage6. Here, we present an apical-out NEC-in-a-dish model, using apical-out enteroids in combination with hypoxia (an important clinical feature of NEC6) and either LPS or TNF-&#945;, as an improved and more physiologically relevant in vitro model to study epithelial responses to NEC-like inflammation and, potentially, identify therapeutic targets. We describe a protocol for reversing the polarity of small intestinal 3D enteroids, as well as an immunofluorescent staining protocol to identify epithelial barrier disruption and junctional protein expression alterations. Finally, we further demonstrate a simple enteroid viability assay to determine the impact of our dual-hit, apical-out NEC-in-a-dish model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Fisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment 24-Well Microplates</td>
			<td>Fisher Scientific</td>
			<td>07-200-602</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass 24 mm x 60 mm</td>
			<td>Thermo Scientific</td>
			<td>102460</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG Antibody, Cy3 conjugate</td>
			<td>Sigma-Aldrich</td>
			<td>AP182C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>E-cadherin antibody (7H12)</td>
			<td>Novus Biologicals</td>
			<td>NBP2-19051</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4%, buffered, pH 6.9</td>
			<td>Millipore Sigma</td>
			<td>1004960700</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Fiji</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>STEMCELL Technologies</td>
			<td>06010</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 Confocal Microscope</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography</td>
			<td>Millipore Sigma</td>
			<td>L3012-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Sigma-Aldrich</td>
			<td>566460</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>Thermo Scientific</td>
			<td>136101</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline), 1x [-] calcium, magnesium, pH 7.4</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Glass Antifade Mountant with NucBlue</td>
			<td>Fisher Scientific</td>
			<td>P36983</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-Villin antibody [SP145]</td>
			<td>Abcam</td>
			<td>ab130751</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-&#945; protein 100 &#181;g</td>
			<td>Bio-Techne</td>
			<td>210-TA-100/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax iD3 Multi-Mode Microplate Reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Forma Series II Water-Jacketed Tri-Gas Incubator, 184L</td>
			<td>Fisher Scientific</td>
			<td>3140</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3 Iodide (642/661)</td>
			<td>Thermo Scientific</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes, 0.5 mL, flat cap</td>
			<td>Thermo Scientific</td>
			<td>AB0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro apical-out enteroid model of necrotizing enterocolitis

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

The use of enteroids to model intestinal inflammation, even within the context of necrotizing enterocolitis, is now common. However, most methods currently utilized either lack access to the apical surface of enteroids, negating the physiological relevance of compounds intended for eventual use as oral therapeutics, or are technically difficult and time-consuming, as with enteroid-derived monolayers. To increase the utility of current in vitro enteroid models of NEC, we reversed the polarity of enteroids, and, i...

Watch this Scientific Journal Video about In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis at JoVE.com

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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

This protocol describes the first apical-out in-a-dish model and is a critical improvement over basal lateral out enteroid in-a-dish models when establishing in vitro modeling of potential therapeutics intended for oral administration. This method allows access to the apical surface of enteroids. Compounds can be added to cell media and the compounds are taken up apically as they would be in vivo.Investigators interested in establishing an in vitro system of intestinal inflammation to test potential oral therapeutics may find this technique especially useful. Tissue sourced from different ages and species may require further standardization. So patients while establishing polarity reversal times may be required.For reversing the polarity of 3D enteroids aspirate media from each well of a 24 well plate of established 3D basolateral out enteroids. Add 500 microliters of five millimolar ice cold, EDTA or PBS to each well of a 24 well plate and gently mechanically disrupt the basement membrane extract or extracellular matrix dome by pipetting up and down five to six times with a P 200 pipette. Transfer the contents of four wells to a 15 milliliter chronicled tube.Then add eight milliliters of ice cold EDTA slash PBS to the tube. Transfer the remaining 20 wells in the same manner. Incubate the tubes at four degrees Celsius for one hour on a rotating platform or shaker at 330 RPM.After incubation centrifuge the tubes and discard the supernatant from each tube. Combine and wash the pellets with five milliliters of DMEM F12. Then centrifuge the cells suspension and remove the supernatant before adding 12 milliliters of antibiotic free medium to the tube ensuring that the cell pellets are resuspended.Pippet 500 microliters of the enteroid suspension into each well of a 24 well ultra-low attachment plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for two to five days or until the enteroids have reversed polarity. On day one abstaining, transfer the contents of four wells of a 24 well plate to a 1.5 milliliter micro centrifuge tube.Repeat for all desired wells with each treatment in a separate tube. Centrifuge the tubes and resuspend the pellet in 300 microliters of 4%from aldehyde fixative at room temperature. After 30 minutes wash the pellets with 500 microliters of PBS.Add 500 microliters of 0.1%Triton X 100 to the tubes. Incubate the tubes for one hour at room temperature. Next, place the tubes on a rotator or shaker at 200 RPM for 15 minutes at two to eight degrees Celsius.After the last incubation, add 500 microliters of 10%NDS in PBS T and incubate at room temperature for 45 minutes. During incubation, prepare primary antibody solution. The next day add 250 to 500 microliters PBS T to the tubes depending on the size of the pellet, and place them on the rotator or shaker at 200 RPM for one hour at two to eight degrees Celsius.Add 200 microliters of the secondary antibody solution and incubate the tubes at two to eight degrees Celsius in the dark. The next day, proceed for mounting stained apical out enteroids by spinning the tubes and removing 100 microliters of supernatant. Resuspend the cells in the remaining 100 microliters of supernatant and transfer the cell suspension to 500 microliter tubes.Centrifuge the tubes in a mini centrifuge for 20 seconds. Remove the supernatant and resuspend the pellets in 100 microliters of room temperature far red nucleic acid stain. After 20 minutes of incubation, centrifuge the cells and resuspend the pellets in 100 microliters of PBS.After the second wash and centrifugation remove 70 microliters of the supernatant and resuspend the pellets in the remaining volume of PBS. Then transfer the cell suspension to a labeled 24 by 60 millimeter cover slip. Apply 75 microliters of mountant directly onto the specimen and remove any bubbles with a pipette tip.After overnight curing in the dark, apply a thin layer of glycerol to the cover slips, then mount the cover slips onto the glass slide and gently press it into place. Tap to remove any bubbles between the cover slip and the slide. Allow the cover slip to set at room temperature in the dark for two hours before imaging the enteroids at 20 times magnification with a confocal microscope.For enteroid treatments, establish an apical out necrotizing enterocolitis in a dish model for 24 hours as described in the text manuscript. Before performing the assay, remove 100 microliters of media from each well leaving the remaining 400 microliters. Add 400 microliters of cell viability assay reagent to each well.Vigorously mix the contents on a plate shaker for five minutes at 200 RPM to induce cell lysis. Next, transfer 200 microliters to a single well of a 96 well clear bottomed plate. Repeat for the remaining 600 microliters creating four technical replicates per well.Using a plate reader capable of luminescence, record values at 0.25 milliseconds integration and compare the relative values among treatments. The representative immunofluorescence staining of control confirmed the apical out localization of nuclei toward the lumen and the detection of villain at the outer edge of the epithelium. Compared to the control, exposure to lipo polysaccharide, tumor necrosis alpha, or hypoxia alone did not induce overt changes in gross cell morphology E-cadherin or villain localization or fluorescent intensity.Treatment with lipo polysaccharide or tumor necrosis alpha in combination with hypoxia disrupted epithelial architecture and demonstrated a significant loss of adherence junction and brush border protein expression revealed by the loss of ECA hearing and villain staining. The apical out cell viability was determined under normoxic or hypoxic conditions. Under normoxic conditions compared to the control lipo polysaccharide or tumor necrosis alpha did not significantly affect the cell viability of enteroids.However, significant reductions in viability were observed in lipo polysaccharide or tumor necrosis alpha in combination with hypoxia compared to hypoxia alone. While establishing apical out in-a-dish model. Remember to keep EDTA in the cell suspension ice cold.This will enhance polarity reversal and ensure all ECM is properly liquified and removed. Other downstream applications such as QPCR, RNA-seq, western blotting or functional evaluations of barrier permeability can be performed further to characterize the response to inflammatory signaling in hypoxia.

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