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. Live-cell imaging services provided by the Cancer Functional Genomics core were supported partly by the National Institute of General Medical Sciences Grant P20GM103639 and National Cancer Institute Grant P30CA225520 of the National Institutes of Health, awarded to the University of Oklahoma Health Sciences Center Stephenson Cancer Center.

All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. Following institutional approval, fetal 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)20.
1. Establishment of preterm non-human primate 3D intestinal entero...

<ol>
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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=Very+low+birth+weight+outcomes+of+the+National+Institute+of+Child+Health+and+Human+Development+Neonatal+Research+Network,+January+1995+through+December+1996.">Very low birth weight outcomes of the National Institute of Child Health and Human Development Neonatal Research Network, January 1995 through December 1996.</a> Pediatrics. 107 (1), 1 (2001).</li><li>Burge, K., Vieira, F., Eckert, J., Chaaban, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+composition,+digestion,+and+absorption+differences+among+neonatal+feeding+strategies:+Potential+implications+for+intestinal+inflammation+in+preterm+infants.">Lipid composition, digestion, and absorption differences among neonatal feeding strategies: Potential implications for intestinal inflammation in preterm infants.</a> Nutrients. 13 (2), 550 (2021).</li><li>Duffy, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+mediating+bacterial+translocation+in+the+immature+intestine.">Interactions mediating bacterial translocation in the immature intestine.</a> The Journal of Nutrition. 130, 432-436 (2000).</li><li>Nanthakumar, N., 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=The+mechanism+of+excessive+intestinal+inflammation+in+necrotizing+enterocolitis:+An+immature+innate+immune+response.">The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: An immature innate immune response.</a> PLoS One. 6 (3), 17776 (2011).</li><li>Nanthakumar, N. N., Fusunyan, R. D., Sanderson, I., Walker, W. 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R., 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+milk+hyaluronan+enhances+innate+defense+of+the+intestinal+epithelium.">Human milk hyaluronan enhances innate defense of the intestinal epithelium.</a> Journal of Biological Chemistry. 288 (40), 29090-29104 (2013).</li><li>Burge, K., Bergner, E., Gunasekaran, A., Eckert, J., Chaaban, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glycosaminoglycans+in+protection+from+neonatal+necrotizing+enterocolitis:+A+narrative+review.">The role of glycosaminoglycans in protection from neonatal necrotizing enterocolitis: A narrative review.</a> Nutrients. 12 (2), 546 (2020).</li><li>Chaaban, H., 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=Acceleration+of+small+intestine+development+and+remodeling+of+the+microbiome+following+hyaluronan+35+kDa+treatment+in+neonatal+mice.">Acceleration of small intestine development and remodeling of the microbiome following hyaluronan 35 kDa treatment in neonatal mice.</a> Nutrients. 13 (6), 2030 (2021).</li><li>Gunasekaran, 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=Hyaluronan+35+kDa+enhances+epithelial+barrier+function+and+protects+against+the+development+of+murine+necrotizing+enterocolitis.">Hyaluronan 35 kDa enhances epithelial barrier function and protects against the development of murine necrotizing enterocolitis.</a> Pediatric Research. 87 (7), 1177-1184 (2020).</li><li>Montenegro-Miranda, P. 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The gastrointestinal tract of a preterm infant is under continual regenerative pressure from repeated exposures to environmental insults associated with dysbiosis, inflammatory bacterial metabolites and toxins, and intermittent hypoxia23,24. Unfortunately, the intestinal epithelium of the preterm infant is unable to rapidly establish functional integrity23, resulting in barrier dysfunction, increased intestinal permeability, and, in severe...

Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in preterm infants1. The disease is characterized by severe intestinal inflammation that can rapidly deteriorate to intestinal necrosis, sepsis, and potentially death. Although the etiology is unclear, evidence suggests NEC is multifactorial and the result of a complex interaction of feeding, abnormal bacterial colonization, and an immature intestinal epithelium2,3. Preterm infants have increased intestinal permeability, abnormal bacterial colonization, and low enterocyte regenerative capacity4,5, increasing their risk for intestinal barrier dysfunction, bacterial translocation, and NEC development. Therefore, identifying strategies or interventions to accelerate intestinal epithelial maturation and promote regeneration or healing of the intestinal epithelium is critical in preventing this deadly disease.
Studies have demonstrated that human milk (HM) is protective against NEC in preterm infants6,7,8,9,10,11. Both human and animal studies have shown that bovine-based formula increases intestinal permeability and is directly toxic to intestinal epithelial cells2,12. Although not fully elucidated, evidence suggests the protective effects of HM are mediated through bioactive components such as lactoferrin, immunoglobulin A (IgA), and HM oligosaccharides13. HM is also rich in hyaluronan (HA), a uniquely nonsulfated glycosaminoglycan with repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharides14,15. Importantly, we have shown that oral 35 kDa HA (HA35), an HM HA mimic, attenuates the severity of intestinal injury, prevents bacterial translocation, and decreases mortality in a murine NEC-like intestinal injury model16,17.
Here, the effects of HA35 on intestinal healing and regeneration in vitro are further investigated. Currently, the most widely used in vitro assay for intestinal wounding and repair is a scratch wound assay performed in colorectal cancer (CRC) cell monolayers. The physiological relevance of such a model to the preterm infant intestine is limited, as wound repair of CRC cells relies heavily upon the highly proliferative nature of cancer cells rather than stem cell-driven repair processes18. To overcome this limitation, the establishment of a 2D enteroid scratch wound model, including the procedure of isolating and maintaining primary stem cell-derived small intestinal enteroids from preterm non-human primates (NHP), is described here. Given preterm NEC is most often reported in the distal small intestine, the use of primary epithelial cell organoids in a model of intestinal damage and repair provides a more physiologically translatable in vitro model compared with existing models utilizing traditional colorectal monolayers18,19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipet</td>
			<td>Fisher Scientific</td>
			<td>13-675-49</td>
			<td></td>
		</tr>
		<tr>
			<td>100x21mm Dish, Nunclon Delta</td>
			<td>ThermoFisher Scientific</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well, TC-Treated, Flat Bottom Plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;M Reversible Cell Strainer</td>
			<td>STEMCELL Technologies</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Sterile Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>FB22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</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>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>100-0485</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte S3 Live-Cell Analysis Instrument</td>
			<td>Sartorius</td>
			<td>4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Scratch Wound Analysis Software Module</td>
			<td>Sartorius</td>
			<td>9600-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>STEMCELL Technologies</td>
			<td>06010</td>
			<td>This is HOGMY, but without the Y-27632 or antibiotics. Also used as base for HOGM, but then only missing the antibiotics.</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>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>ThermoFisher 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>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>This is broad-spectrum antibiotics</td>
		</tr>
		<tr>
			<td>Sodium Hyaluronate, Research Grade, HA20K</td>
			<td>Lifecore Biomedical</td>
			<td>HA20K-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Company: Bio-Rad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1X, 0.25% Trypsin</td>
			<td>Fisher Scientific</td>
			<td>MT25053CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of hyaluronic acid 35 kda on an in vitro model of preterm small intestinal injury and healing using enteroid-derived monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

The effects of HA on tissue repair and wound healing in various tissues and organs are well-documented; however, the specific effects of HA with a molecular weight of 35 kDa on fetal or neonatal small intestinal healing and regeneration are currently unknown. To test the ability of HA35 to promote wound healing in a model of the fetal or neonatal small intestine, we generated 3D intestinal enteroids from NHP ileal tissue and further dissociated this tissue into single cells to create 2D enteroid-derived monolayers (<stro...

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Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

This protocol extends the very common scratch wound assay used for regeneration studies with enteroid-derived monolayers representing all major cell types of the small intestine. This technique enables real-time visualization of wound closure in a preterm 2D enteroid monolayer model of intestinal regeneration. Our technique can be applied to the study of preterm mammalian gut, which is typically poorly modeled using transformed colorectal cancer cell lines.Begin by thawing 96 microliters of extracellular matrix, or ECM-based hydrogel BME, overnight on ice at two to eight degrees Celsius. Dilute the ECM-based hydrogel and ice-cold PBS without calcium or magnesium at a ratio of one to 50. Coat each well of a 24-well tissue culture plate with 200 microliters of ice-cold diluted ECM-based hydrogel and incubate the coated plate for a minimum of one hour at 37 degrees Celsius and 5%carbon dioxide.Then aspirate the media from the 24-well culture plate containing the 3D enteroids destined for monolayers and replace it with 500 microliters of cell dissociation reagent per well. Manually disrupt the ECM-based hydrogel domes by pipetting up and down eight to 12 times and transferring the contents of 12 wells to a 15-milliliter conical tube. Wash the 12 empty wells with 250 microliters of cell dissociation reagent to ensure all the enteroids have been removed and transfer the contents to 15-milliliter conical tubes.Centrifuge the conical tubes at 300g for five minutes at four degrees Celsius and discard the supernatant. Add 10 milliliters of ice-cold DMEM/F-12 wash buffer to the conical tubes and suspend the enteroid pellets by inverting the tubes. Centrifuge at 300g for five minutes at four degree Celsius and remove the supernatant.Resuspend the cell pellets in 12 milliliters of 37 degrees Celsius Trypsin-EDTA and place the tubes in a 37 degree Celsius water bath for 10 minutes or until the cells appear soluble. Add three milliliters of DMEM/F-12 wash buffer to each tube to dilute Trypsin-EDTA. Mix by inverting the tubes and place on ice.Filter the dissociated enteroids through a 37 micrometer mesh cell strainer. Collect the contents into clean 15-milliliter conical tubes and centrifuge the tubes at 300g for five minutes at four degrees Celsius. Remove the supernatant from the conical tubes and resuspend the cell pellets with six milliliters of HOMGY.Next, remove the ECM-based hydrogel coated plate from the incubator and aspirate any excess ECM-based hydrogel solution without scratching the coated surface or completely drying the plate. Add 500 microliters of the combined 12 milliliters HOMGY cell suspension into each well of the 24-well plate coated with ECM-based hydrogel. Swirl the plate with the lid on to ensure an even distribution of cells within the wells.Incubate the plate at 37 degrees Celsius and 5%carbon dioxide, exchanging HOMGY media every two days until the monolayers reach greater than 90%confluency. Once the monolayers reach greater than 90%confluency, aspirate the media to remove any cells failing to attach. Treat the cells with 500 microliters of HA35 or PBS dissolved in HOMGY for 24 hours.After that, remove the media without disrupting the surface of the monolayer. Using a 200-microliter pipette tip, make a linear scratch across the full surface diameter of the monolayer in each well, being careful not to let the monolayers dry. Wash the scratched monolayers once with 500 microliters of 1X PBS and add 500 microliters of HA35 or PBS dissolved in HOMGY.Transfer the plate to the live cell analysis instrument within a 37 degree Celsius and 5%carbon dioxide incubator. Under the Schedule icon in the live cell analysis software, click on the Launch Add New Vessel Wizard icon. Select Scan on Schedule, then click on Next.Select New under Create Vessel and click on Next, then select Whole Well under Scan Type and click Next. Choose Phase for image channels and 4x for the objective. Select the plate manufacturer and the catalog number from the provided list and click Next.Select an empty vessel location followed by the desired scan pattern. Enter plate name in Name, then select plus to create a plate layout. Select small plus to create a treatment name.Enter short name and then OK.Highlight the plate wells corresponding to that treatment and hit large plus to designate wells with that treatment. Enter compound concentration and click OK.When plate is complete, select OK, Next. Select Defer analysis until later followed by Create new schedule with scans at intervals of four hours.Stop scanning at one days, zero hours. Ensure that the summary is correct and then add to schedule. In live cell analysis software, select View at the top of the screen.From the Vessel Name menu, navigate to your completed 24 hour scan and double-click the vessel name. Select Export images and movies icon on left followed by As Displayed option. Highlight the wells of interest, and then select time points of interest and Series of Images as Displayed.Ensure the sequence type and the scans are correct. Select desired target folder location. Choose JPEG from the dropdown menu and then Export.Open ImageJ software and upload the scratch wound assay image. Select Image, then type 8-bit to change the image from 24-bit RGB. Select Plugins, Macros, Install, Wound_healing_size plugin to install the Wound Healing Size Plugin.After that, set the variance window radius to 20, threshold value to 100, percentage of saturated pixels to 0.001. Yes for Set Scale global and then click on OK.Verify that the outlined area is the wound area and repeat for additional scratches. Finally, calculate the percentage of the scratch area wound healing of migrating or proliferating cells over 24 hours using the equation shown on the screen.Here, T0 is the area at time zero hours, and TC is the area at zero hours, four hours, 12 hours, or 24 hours. Enteroid generation and enteroid-derived monolayer wound healing assay procedure are shown in these images. A culture of three-dimensional, non-human primate intestinal enteroids is used to dissociate into two-dimensional monolayers.Monolayers were grown to greater than 90%confluence in a 24-well plate and then treated with HA35 or PBS for 24 hours. Monolayers were then scratched with a P200 pipette tip. Shown here are the representative images of HA35 in control treatments of nonhuman primate enteroid monolayers.Images were obtained at zero hours, four hours, 12 hours, and 24 hours after performing a scratch with a P200 pipette tip. The effect of HA35 on wound healing andoid monolayers is shown here. The change in wound healing over time in enteroid monolayers is dependent on HA35 treatment concentration.HA35 significantly increased wound healing after four hours and 12 hours at 100 micrograms per milliliter and 200 micrograms per milliliter when compared to control, but healing rates converged among treatments by 24 hours. One of the most important things to remember with this procedure is that intestinal enteroid monolayers are short-lived. So, once they're 90%confluid, investigators should look to use them quickly.This technique has become our go-to model for preterm intestinal regeneration in vitro, especially if we are interested in more mechanical rather than inflammatory injury.

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2.1K vistas.  University of Oklahoma Health Sciences Center. Este protocolo amplía el ensayo muy común de herida por rasguño utilizado para estudios de regeneración con monocapas derivadas de enteroides que representan todos los tipos principales de células del intestino delgado. Esta técnica permite la visualización en tiempo real del cierre de la herida en un modelo monocapa enteroide 2D prematuro de regeneración intestinal. Nuestra técnica se puede aplicar al estudio del intestino prematuro de mamíferos, que generalmente está mal modelado...