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 enteroids
<ol>
	<li>Media preparation 
	NOTE: Media can be prepared up to 1 week in advance of crypt isolation following standard aseptic technique. Penicillin/streptomycin (final concentration 1%) may be used in place of the broad-spectrum antibiotics for primary cells if desired.
	<ol>
		<li>Prepare human organoid growth media + Y-27632 (HOGMY) by mixing 50 mL of organoid growth medium human basal medium with 50 mL of organoid supplement (Table of Materials). Add 200 &#181;L of the broad-spectrum antibiotics (final concentration 100 &#181;g/mL) (Table of Materials) and Y-27632 (to a final concentration of 10 &#181;M). Warm on the benchtop to room temperature.</li>
		<li>Prepare human organoid growth media (HOGM) by mixing 50 mL of organoid growth medium human basal medium with 50 mL of organoid supplement. Add 200 &#181;L of the broad-spectrum antibiotics (100 &#181;g/mL). Warm on the benchtop to room temperature. 
		NOTE: HOGM will be used when changing media beyond the first 2-3 days post-passage.</li>
		<li>Chill 100 mL of 1x phosphate-buffered saline (PBS) without calcium or magnesium until ice-cold.</li>
		<li>Prepare 50 mL of wash buffer by combining 1x PBS without calcium or magnesium with 0.1% bovine serum albumin (BSA). Chill it until ice-cold.</li>
		<li>Prepare DMEM-F12 wash buffer by adding 1 mL of the broad-spectrum antibiotics (100 &#181;g/mL) to 500 mL of DMEM/F12 (Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F12 + 15 mM HEPES buffer). Keep it ice-cold.</li>
	</ol>
	</li>
	<li>Crypt isolation and plating 
	NOTE: Extracellular matrix (ECM)-based hydrogel must remain on the ice during the entire procedure. This procedure assumes enough tissue to populate six ECM-based hydrogel domes. If a different density of crypts is harvested, the dome number should be altered accordingly.
	<ol>
		<li>Thaw 150 &#181;L of growth factor reduced (GFR) basement membrane extract (BME) (phenol red-free) overnight on the ice at 2-8 &#176;C.</li>
		<li>Incubate a 24-well, flat-bottom, tissue culture-treated polystyrene plate in an incubator at 37 &#176;C and 5% CO2 for a minimum of 30 min before use.</li>
		<li>Following NHP euthanasia and tissue collection, flush the ileum segment of debris with a 1,000 &#181;L pipette tip using ice-cold 1x PBS in a disposable Petri dish. 
		NOTE: Fresh tissue must be harvested quickly and kept ice-cold to harvest healthy enteroids.</li>
		<li>Cut the ileum longitudinally along the entire intestine, and wash with ice-cold PBS 3x. Mince the tissue into small fragments (approximately 2 mm in length) with sterile scissors over a 50 mL conical tube containing 15 mL ice-cold PBS, starting from the portion of the ileal segment closest to the tube.</li>
		<li>Use a 10 mL serological pipette to disrupt the crypts by pipetting up and down 5-10x. Let the tissue segments settle to the bottom of the tube, then remove the supernatant and replace it with fresh, ice-cold PBS. Repeat this process a minimum of 7x-10x until the supernatant is clear and free of debris.</li>
		<li>Once clear, remove the supernatant and resuspend the crypts in 25 mL of cell dissociation reagent. Place the tube on a rocking platform at 20 rpm for 15 min at room temperature.</li>
		<li>Remove the tube from the rocker, allow the cells to settle for approximately 30 s to 1 min, and remove the supernatant. Add 10 mL of ice-cold wash buffer. Pipette up and down with a p1000 pipette to further dissociate into single crypts.</li>
		<li>Filter the tissue through a 70 &#181;m cell strainer into a new 50 mL conical tube. Rinse the original 50 mL conical tube with 10 mL of ice-cold wash buffer and filter through the 70 &#181;m strainer to ensure all the crypts have been removed from the original tube. Repeat this washing of tissue for a total of 4x rinses.</li>
		<li>Centrifuge the filtrate at 300 x g for 5 min at 4 &#176;C and remove the supernatant. Add 600 &#181;L of HOGMY and disrupt the pellet by pipetting up and down with a 200 &#181;L pipette. Place the tube on ice until the solution becomes ice-cold. 
		NOTE: If the solution is not ice-cold, the dome will not maintain the proper structure when plated. Avoid introducing bubbles when pipetting ECM-based hydrogel&#160;domes, as bubbles will prevent proper adherence to the plate bottom.</li>
		<li>Add 600 &#181;L of ice-cold ECM-based hydrogel to suspend the cell pellet and mix well with a 200 &#181;L pipette.</li>
		<li>Remove the 24-well plate from the incubator and place it on a 37 &#176;C plate warmer. Pipette 50 &#181;L of ice-cold HOGMY/ECM-based hydrogel mixture into the center of 24 wells of a 24-well culture plate (50 &#181;L per dome).</li>
		<li>Once plated, let the domes sit on a 37 &#176;C plate warmer for 1 min to allow adherence to the bottom of the plate. Carefully invert the plate, ensure no condensation has accumulated on the bottom of the plate, and place the inverted plate in the incubator at 37 &#176;C and 5% CO2.</li>
		<li>After 15 min, turn the plate right-side-up and pipette 750 &#181;L of room-temperature HOGMY into each well. 
		NOTE: Pipette media slowly onto the sidewall of wells, being careful not to disrupt the newly formed 3D ECM-based hydrogel dome.</li>
		<li>Once all the wells have been filled with media, place the plate in the incubator and change the media every 3 days with 750 &#181;L of HOGM. Passage enteroids every 7-10 days.</li>
	</ol>
	</li>
	<li>Intestinal enteroid passaging 
	NOTE: The following protocol is for one 24-well culture plate containing NHP enteroids at a split rate of 1:2.
	<ol>
		<li>Remove the enteroid media from each well, being careful not to disrupt the ECM dome containing enteroids, add 500 &#181;L of ice-cold cell dissociation reagent to each well using a 1,000 &#181;L pipette, and incubate at room temperature for 1 min.</li>
		<li>Manually disrupt the ECM by pipetting up and down 8-12x. Transfer the contents of 12 wells to one 15 mL conical tube.</li>
		<li>Once all the solution has been collected from the 24-well culture plate, add 250 &#181;L of cell disruption reagent to each well to ensure all the NHP enteroids have been removed from each well, adding this last rinse to the existing 15 mL conical tubes.</li>
		<li>Place on a rocking platform at 40 rpm for 15 min at room temperature (20 &#176;C). Alternatively, manually shake the tubes for 15 min at room temperature.</li>
		<li>After 15 min, centrifuge at 300 x g for 5 min at 4 &#176;C. Remove the supernatant from the 15 mL tubes until only the pellet remains.</li>
		<li>Add 8 mL of ice-cold DMEM + broad-spectrum antibiotics to one 15 mL conical tube, making sure the pellet is sufficiently disrupted. Centrifuge at 300 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Add 600 &#181;L of HOGMY and disrupt the pellet by pipetting up and down with a 1,000 &#181;L pipette. Place the tube on ice until the solution becomes ice-cold. Once the solution is ice-cold, add 600 &#181;L of ice-cold ECM to suspend the cell pellet and mix well with a 1,000 &#181;L pipette. Repeat steps 1.3.2.-1.3.7. for the remaining 12 wells.
		<ol>
			<li>For the remaining steps, refer to steps 1.2.11.-1.2.16.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Establishment of enteroid monolayer and scratch wound assay
<ol>
	<li>Media and treatment preparation
	<ol>
		<li>Chill 5 mL of PBS without calcium or magnesium until ice-cold.</li>
		<li>Prepare DMEM-F12 wash buffer by adding 1 mL of the broad-spectrum antibiotics (100 &#181;g/mL) to 500 mL of DMEM/F12 (Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F12 + 15 mM HEPES buffer). Keep ice-cold.</li>
		<li>Prepare 100 mL of HOGMY, as described in step 1.1.1., and warm it on the benchtop to room temperature.</li>
		<li>Warm 25 mL of 0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA) in a 37 &#176;C water bath.</li>
		<li>Prepare 50 &#181;g/mL, 100 &#181;g/mL, and 200 &#181;g/mL concentrations of HA35 for cell treatment, using sterile PBS without calcium or magnesium as a solvent. The final dilution of HA35 will use HOMGY as the solvent. 
		NOTE: HA35 preparation may require several dilutions, as HA35 often comes out of solution at a concentration greater than 5 mg/mL.</li>
	</ol>
	</li>
	<li>Enteroid monolayer formation 
	NOTE: The following procedure provides volumes sufficient for the generation of one 24-well plate of enteroid monolayers. Adjust the volumes for the desired number of monolayers.
	<ol>
		<li>Thaw 96 &#181;L of ECM-based hydrogel BME (phenol red-free) overnight on ice at 2-8 &#176;C. Dilute ECM-based hydrogel in ice-cold PBS without calcium or magnesium at a ratio of 1:50. Coat each well of a 24-well tissue culture plate with 200 &#181;L of ice-cold diluted ECM-based hydrogel.</li>
		<li>Incubate the ECM-based hydrogel-coated plate for a minimum of 1 h at 37 &#176;C and 5% CO2. Aspirate media from the 24-well culture plate containing the 3D enteroids destined for monolayers and replace with 500 &#181;L of cell dissociation reagent/well.</li>
		<li>Manually disrupt ECM-based hydrogel domes by pipetting up and down 8x-12x and transfer the contents of 12 wells to a 15 mL conical tube.</li>
		<li>Wash the 12 empty wells with 250 &#181;L of cell dissociation reagent to ensure all the enteroids have been removed and transfer the contents to a 15 mL conical tube. Repeat step 2.2.5. and step 2.2.6. for the remaining 12 wells using a second 15 mL conical tube.</li>
		<li>Centrifuge the conical tubes at 300 x g for 5 min at 4 &#176;C and discard the supernatant. Add 10 mL of ice-cold DMEM-F12 wash buffer to 15 mL conical tubes and suspend the enteroid pellets by inverting the tubes. Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend the cell pellets in 12 mL of 37 &#176;C Trypsin-EDTA. Place the tubes in a 37 &#176;C water bath for 10 min or until the cells appear soluble. Add 3 mL of DMEM-F12 wash buffer to each tube to dilute Trypsin-EDTA, mix by inverting the tubes, and place on ice.</li>
		<li>Filter the dissociated enteroids through a 37 &#181;M mesh cell strainer, collecting the contents into clean 15 mL conical tubes. Centrifuge the tubes at 300 x g for 5 min at 4 &#176;C.</li>
		<li>While the cells are pelleting in the centrifuge, remove the ECM-based hydrogel-coated plate from the incubator and aspirate any excess ECM-based hydrogel solution without scratching the coated surface or letting the plate completely dry.</li>
		<li>Remove the supernatant from the conical tubes and resuspend the cell pellets with 6 mL of HOMGY. Add 500 &#181;L of the combined 12 mL HOMGY cell suspension into each well of the 24-well plate coated with ECM-based hydrogel at a seeding density of roughly 3 x 105 cells/well. Swirl the plate with a lid on to ensure an even distribution of cells within the wells.</li>
		<li>Incubate the plate at 37 &#176;C and 5% CO2, exchanging HOMGY media every 2 days until the monolayers reach &#62;90% confluency.</li>
	</ol>
	</li>
	<li>Monolayer treatment and scratch wound assay
	<ol>
		<li>Once the monolayers reach &#62;90% confluency, aspirate media to remove any cells failing to attach. Treat cells with 500 &#181;L of HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or PBS, dissolved in HOMGY, for 24 h.</li>
		<li>After 24 h, remove the media, being careful not to disrupt the surface of the monolayer. Using a 200 &#181;L pipette tip, make a linear scratch across the full surface diameter of the monolayer in each well, being careful not to let monolayers dry.</li>
		<li>Wash the scratched monolayers 1x with 500 &#181;L of 1x PBS. Add 500 &#181;L of HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or PBS dissolved in HOMGY and transfer the plate to the live-cell analysis instrument (Table of Materials) within a 37 &#176;C and 5% CO2 incubator.</li>
		<li>Under the Schedule icon in the live-cell analysis software (Table of Materials), click on the Launch Add New Vessel Wizard icon and set the scanning frequency to scan on a schedule. Create a new Whole Well vessel under Scan Type, specify the scan settings (i.e., Phase for Image Channels and 4x for Objective), and click on Next.</li>
		<li>Select the plate manufacturer and catalog number from the provided list. Under the Schedule icon, choose the plus symbol where the plate will be placed within the live-cell analysis instrument. Select the&#160;desired Scan Pattern, and then create a plate map by selecting&#160;&#34;+&#34; on the&#160;Plate Layout&#160;page. Select Defer Analysis Until Later, and then set the Scanning Schedule for every 4 h for 24 h and verify acquisition settings under the Vessel Wizard Summary page. Finally, select&#160;Add to Schedule.</li>
		<li>Once the 24 assay is complete, under the View icon, double-click Vessel Name, and export images and movies. Select wells and a series of images to display and export as a JPEG. Analyze images for percent wound healing using the Wound Healing Size Plugin for ImageJ21, as per step 2.4. below.</li>
	</ol>
	</li>
	<li>Scratch wound analysis
	<ol>
		<li>Open ImageJ software. Upload the scratch wound assay images (.TIFF or .JPG).</li>
		<li>Select Image &#62; Type &#62; 8-bit to change the image from 24-bit RGB to 8-bit. Install the Wound Healing Size Plugin by selecting Plugins &#62; Macros &#62; Install &#62; Wound Healing Size Plugin.</li>
		<li>Rotate the image so the scratch is vertical and initialize the Wound Healing Size Tool plugin.</li>
		<li>Select the plugin parameters in the dialog box to best fit the scratch wound. Set the value of the parameters for wound healing size options as follows: Variance Window Radius at 20, Threshold value at 100, and Percentage of Saturated Pixels at 0.001, and choose Yes for Set Scale Global. Finalize the selection by clicking on the OK button. Verify if the selected area is the wound area. The above parameters may require standardization from lab to lab.</li>
		<li>Repeat steps 2.4.2.-2.4.4. for the remaining time points for each scratch.</li>
		<li>Calculate the percentage of scratch area wound healing of migrating or proliferating cells over 24 h as follows: 
		%&#160;Wound Healing = <img alt="Equation 1" src="/files/ftp_upload/63758/63758eq01.jpg" style="margin: auto;" /> 
		where T0 = % Area at time 0 h and Tc = % Area at 0 h, 4 h, 12 h, or 24 h.</li>
	</ol>
	</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+role+of+35+kilodalton+hyaluronan+in+promoting+defense+of+the+intestinal+epithelium.">Multifunctional role of 35 kilodalton hyaluronan in promoting defense of the intestinal epithelium.</a> The Journal of Histochemistry and Cytochemistry. 66 (4), 273-287 (2018).</li><li>Fraser, J. R. E., Laurent, T. C., Laurent, U. B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan:+Its+nature,+distribution,+functions+and+turnover.">Hyaluronan: Its nature, distribution, functions and turnover.</a> Journal of Internal Medicine. 242 (1), 27-33 (1997).</li><li>Kim, Y., 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+35kDa+treatment+protects+mice+from+Citrobacter+rodentium+infection+and+induces+epithelial+tight+junction+protein+ZO-1+in+vivo.">Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.</a> Matrix Biology. 62, 28-39 (2017).</li><li>Prehm, P., Schumacher, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+hyaluronan+export+from+human+fibroblasts+by+inhibitors+of+multidrug+resistance+transporters.">Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters.</a> Biochemical Pharmacology. 68 (7), 1401-1410 (2004).</li><li>Stenson, W. F., Ciorba, 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=Nonmicrobial+activation+of+TLRs+controls+intestinal+growth,+wound+repair,+and+radioprotection.">Nonmicrobial activation of TLRs controls intestinal growth, wound repair, and radioprotection.</a> Frontiers in Immunology. 11 (3591), 617510 (2021).</li><li>Riehl, T. E., Ee, X., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+regulates+normal+intestinal+and+colonic+growth+in+mice.">Hyaluronic acid regulates normal intestinal and colonic growth in mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 303 (3), 377-388 (2012).</li><li>Riehl, T. E., Santhanam, S., Foster, L., Ciorba, M., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+and+TLR4+mediate+hyaluronic+acid+regulation+of+Lgr5++stem+cell+proliferation,+crypt+fission,+and+intestinal+growth+in+postnatal+and+adult+mice.">CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 309 (11), 874-887 (2015).</li><li>Hill, D. R., Kessler, S. P., Rho, H. K., Cowman, M. K., de la Motte, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific-sized+hyaluronan+fragments+promote+expression+of+human+beta-defensin+2+in+intestinal+epithelium.">Specific-sized hyaluronan fragments promote expression of human beta-defensin 2 in intestinal epithelium.</a> Journal of Biological Chemistry. 287 (36), 30610-30624 (2012).</li><li>Fernando, E. H., Gordon, M. H., Beck, P. L., MacNaughton, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intestinal+epithelial+wound+healing+through+protease-activated+receptor-2+activation+in+Caco2+cells.">Inhibition of intestinal epithelial wound healing through protease-activated receptor-2 activation in Caco2 cells.</a> Journal of Pharmacology and Experimental Therapeutics. 367 (2), 382-392 (2018).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Roodsant, 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=A+human+2D+primary+organoid-derived+epithelial+monolayer+model+to+study+host-pathogen+interaction+in+the+small+intestine.">A human 2D primary organoid-derived epithelial monolayer model to study host-pathogen interaction in the small intestine.</a> Frontiers in Cellular and Infection Microbiology. 10, 272 (2020).</li><li>Singh, A., Poling, H. M., Spence, J. R., Wells, J. M., 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=Gastrointestinal+organoids:+a+next-generation+tool+for+modeling+human+development.">Gastrointestinal organoids: a next-generation tool for modeling human development.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 319 (3), 375-381 (2020).</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. 239 (9), 1124-1134 (2014).</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+a+model+of+upper+small+intestinal+ion+transport+physiology+and+pathophysiology.">Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.</a> Gastroenterology. 150 (3), 638-649 (2016).</li></ol>

The authors have nothing to disclose and no conflicts of interest.

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><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 &amp;micro;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 &amp;micro;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&#039;s Modified Eagle&#039;s Medium/Nutrient Ham&#039;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...

Watch this Scientific Journal Video about Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers at JoVE.com

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. ...

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Research

JoVE Journal

Medicine

2.2K Views.  University of Oklahoma Health Sciences Center. Dit protocol breidt de veel voorkomende kraswondtest uit die wordt gebruikt voor regeneratiestudies met enteroïde-afgeleide monolagen die alle belangrijke celtypen van de dunne darm vertegenwoordigen. Deze techniek maakt real-time visualisatie van wondsluiting mogelijk in een prematuur 2D enteroïde monolaagmodel van darmregeneratie. Onze techniek kan worden toegepast op de studie van te vroeg geboren darm van zoogdieren, die meestal slecht gemodelleerd is met behulp van getransformeerde col...

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

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Immunology and Infection

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

Bioengineering

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.

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.

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

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Biology

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...

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

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