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The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.
Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein β-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.
Enteroids are three-dimensional (3D) structures derived from organ-restricted human intestinal stem cells1,2. They are made up entirely of epithelial lineage and contain all the differentiated intestinal epithelial cell types2. Enteroids also maintain cellular polarity made up of an apical luminal surface forming an inner compartment and a basolateral surface facing the surrounding media. Enteroids are a unique model in that they preserve the characteristics of the host from which they were generated3. Thus, enteroids generated from premature human infants repres....
The present research was performed in compliance with Institutional Review Board approval (IRB, #11610, 11611) at the University of Oklahoma. Parental consent was required prior to collecting human surgical specimens as per IRB specifications. Following IRB approval and parental consent, human small intestinal tissue was obtained from infants (corrected gestational age (GA) ranging from 36-41 weeks at the time of sample collection, all with a history of preterm birth at an estimated GA of 25-34 weeks, 2:1 M:F) undergoing.......
AO conformation
Enteroids suspended in 50% LWRN media for 72 h assume an AO conformation (Figure 1). This was confirmed via immunofluorescent staining utilizing enteroid whole mounts of the apical protein, zonula occludens-1 (ZO-1), and basolateral protein, β-catenin (Figure 1). AO enteroids show ZO-1 (green) on the outer, apical surface of the enteroid, while β-catenin (red) is on the inner, basolateral surface (
Intestinal permeability is complex and reflective of epithelial barrier function. The intestinal barrier comprises a single layer of epithelial cells that mediates transcellular and paracellular transport14. Paracellular permeability relies on tight junction proteins that seal the space between epithelial cells14. Within this paracellular transport, there are three distinct pathways by which molecules can cross: pore, leak, and unrestricted15. The po.......
We would like to thank Ashley Nelson from the University of Rochester Medical Center for her instrumental help with our enteroid model. We would also like to thank the Division of Pediatric Surgery at the University of Oklahoma for their support of this project. This work was supported by the National Institute of Health [NIH Grant R03 DK117216-01A1], the Oklahoma Center for Adult Stem Cell Research, and the Presbyterian Health Foundation Grant #20180587 awarded to the Department of Surgery at the University of Oklahoma Health Sciences Center.
....Name | Company | Catalog Number | Comments |
[leu] 15-gastrin 1 | Millipore Sigma | G9145-.1MG | |
100 µm sterile cell strainer | Corning | 431752 | |
100% LWRN conditioned media | Made in-house following Miyoshi et al.12 | ||
24-well tissue culture plate | Corning | 3526 | |
96-well black, clear bottom plate | Greiner Bio-One | 655090 | |
A-83-01 | R&D Systems | 2939/10 | |
Alexa Fluor 488 goat anti-rabbit secondary ab, 1:1000 | Invitrogen | A-11034 | |
Alexa Fluor 594 goat anti-mouse secondary ab, 1:1000 | Invitrogen | A-11032 | |
Amphotericin B | Thermo Fisher Scientific | 15290026 | |
Anti-zonula occludens-1 rabbit primary ab, 1:200 | Cell Signaling | #D6L1E | |
Anti-β-catenin mouse primary ab, 1:100 | Cell Signaling | #14-2567-82 | |
B-27 supplement minus Vitamin A | Thermo Fisher Scientific | 17504-044 | |
Barrier PAP pen | Scientific Device Laboratory | 9804-02 | |
BMM (Matrigel) | Corning | CB-40230C | |
Cell Recovery Solution | Corning | 354270 | |
Dissecting scissors | |||
DMEM | Thermo Fisher Scientific | 11-965-118 | |
DMEM/F-12 | Thermo Fisher Scientific | 11320-082 | |
DPBS | Thermo Fisher Scientific | 14-190-144 | |
Epidermal Growth Factor (EGF) | Millipore Sigma | GF144 | |
Ethylenediaminetetraacetic acid (EDTA) | Millipore Sigma | EDS-500G | |
EVOS m7000 Imaging system | Invitrogen | AMF7000 | |
Fetal Bovine Serum (FBS) | Gemini Bio-Products | 100-525 | |
Fluoroshield with DAPI | Millipore Sigma | F6057-20mL | |
Forceps | |||
Gentamicin | Thermo Fisher Scientific | 15-750-060 | |
Glass coverslips | |||
GlutaMAX | Thermo Fisher Scientific | 35050-061 | |
GraphPad Prism 9 | Dotmatics | ||
Insulin | Thermo Fisher Scientific | 12585014 | |
Lipopolysaccharide (LPS) | Millipore Sigma | L2630-25MG | |
Lucifer Yellow CH, Lithium Salt | Invitrogen | L453 | |
Modular incubator chamber | Billups Rothenberg Inc. | MIC101 | |
N-2 supplement | Thermo Fisher Scientific | 17502-048 | |
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) | Thermo Fisher Scientific | 15630-080 | |
N-Acetylcysteine | Millipore Sigma | A9165-5G | |
Nicotinamide | Millipore Sigma | N0636-100G | |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140-148 | |
Refrigerated swinging bucket centrifuge | |||
Refrigerated tabletop microcentrifuge | |||
RPMI 1640 Medium | Thermo Fisher Scientific | 11875093 | |
SB202190 | Millipore Sigma | S7067-5MG | |
SpectraMax iD3 microplate reader | Molecular devices | ||
Tube Revolver Rotator | ThermoFisher Scientific | 88881001 | |
Ultra-low attachment 24-well tissue culture plate | Corning | 3473 | |
Y-27632, ROCK inhibitor (RI) | Tocris | 1254 |
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