Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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 (

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
[leu] 15-gastrin 1Millipore SigmaG9145-.1MG
100 µm sterile cell strainerCorning431752
100% LWRN conditioned mediaMade in-house following Miyoshi et al.12
24-well tissue culture plateCorning3526
96-well black, clear bottom plateGreiner Bio-One655090
A-83-01R&D Systems2939/10
Alexa Fluor 488 goat anti-rabbit secondary ab, 1:1000InvitrogenA-11034
Alexa Fluor 594 goat anti-mouse secondary ab, 1:1000InvitrogenA-11032
Amphotericin BThermo Fisher Scientific15290026
Anti-zonula occludens-1 rabbit primary ab, 1:200Cell Signaling#D6L1E
Anti-β-catenin mouse primary ab, 1:100Cell Signaling#14-2567-82
B-27 supplement minus Vitamin AThermo Fisher Scientific17504-044
Barrier PAP penScientific Device Laboratory9804-02
BMM (Matrigel)CorningCB-40230C
Cell Recovery SolutionCorning354270
Dissecting scissors
DMEMThermo Fisher Scientific11-965-118
DMEM/F-12Thermo Fisher Scientific11320-082
DPBSThermo Fisher Scientific14-190-144
Epidermal Growth Factor (EGF)Millipore SigmaGF144
Ethylenediaminetetraacetic acid (EDTA)Millipore SigmaEDS-500G
EVOS m7000 Imaging systemInvitrogenAMF7000
Fetal Bovine Serum (FBS)Gemini Bio-Products100-525
Fluoroshield with DAPIMillipore SigmaF6057-20mL
Forceps
GentamicinThermo Fisher Scientific15-750-060
Glass coverslips
GlutaMAXThermo Fisher Scientific35050-061
GraphPad Prism 9Dotmatics
InsulinThermo Fisher Scientific12585014
Lipopolysaccharide (LPS)Millipore SigmaL2630-25MG
Lucifer Yellow CH, Lithium SaltInvitrogenL453
Modular incubator chamberBillups Rothenberg Inc.MIC101
N-2 supplementThermo Fisher Scientific17502-048
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)Thermo Fisher Scientific15630-080
N-AcetylcysteineMillipore SigmaA9165-5G
NicotinamideMillipore SigmaN0636-100G
Penicillin-StreptomycinThermo Fisher Scientific15140-148
Refrigerated swinging bucket centrifuge
Refrigerated tabletop microcentrifuge
RPMI 1640 MediumThermo Fisher Scientific11875093
SB202190Millipore SigmaS7067-5MG
SpectraMax iD3 microplate readerMolecular devices
Tube Revolver RotatorThermoFisher Scientific88881001
Ultra-low attachment 24-well tissue culture plateCorning3473
Y-27632, ROCK inhibitor (RI)Tocris1254

References

  1. Ranganathan, S., Smith, E. M., Foulke-Abel, J. D., Barry, E. M. Research in a time of enteroids and organoids: How the human gut model has transformed the study of enteric bacterial pathogens. Gut Microbes. 12 (1), 1795492 (2020).
  2. De Fazio, L., et al.

Explore More Articles

Lucifer YellowApical out Enteroid ModelIntestinal PermeabilityParacellular PathwaysNecrotizing EnterocolitisCell Recovery SolutionCentrifugation3D Model24 well PlateAO Enteroids

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved