Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

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 cells^1,2. 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 surgery for NEC or other intestinal resection, such as ostomy takedown or atresia repair. Enteroids were generated from tissue obtained from either t....

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.......

Materials

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