In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Summary

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.

Abstract

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-α) 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-α for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-α, 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.

Introduction

Necrotizing enterocolitis (NEC), a severe inflammatory disease of the small intestine occurring in up to 10% of preterm infants, is commonly associated with high morbidity and mortality^1,2. Mortality rates approaching 50% in very low birth weight (<1500 g) infants, requiring surgical intervention, are not uncommon³. While the exact etiology of NEC is not currently understood, risk factors, such as formula feeding, are thought to compound with physiological anomalies, such as dysbiosis, an immature intestinal epithelium, and a dysfunctional intestinal barrier, in the development of t....

Protocol

All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. 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).

1. Establishment of apical-out enteroid NEC-in-a-dish model

1. Preparation of media and enteroid treatments

Discussion

The recent development of enteroid models derived from intestinal epithelial crypts allows for a more physiologically relevant in vitro tissue in which to study necrotizing enterocolitis pathogenesis. Despite including all major differentiated cell types of the intestinal epithelium, 3D enteroids are still subject to several significant limitations. The conventional, basolateral-out enteroids are suspended in 3D ECM hydrogel domes, the composition and density of which may limit normal diffusion within the tissue.......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Fisher Scientific	15575-020
1.5 mL microcentrifuge tubes	Fisher Scientific	05-408-129
15 mL Conical tube	VWR	89039-666
CellTiter-Glo 3D Cell Viability Assay	Promega	G9681
Corning Costar Ultra-Low Attachment 24-Well Microplates	Fisher Scientific	07-200-602
Cover Glass 24 mm x 60 mm	Thermo Scientific	102460
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Scientific	A-21202
Donkey Anti-Rabbit IgG Antibody, Cy3 conjugate	Sigma-Aldrich	AP182C
Dulbecco's Modified Eagle's Medium/Nutrient Ham's Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer	STEMCELL Technologies	36254
E-cadherin antibody (7H12)	Novus Biologicals	NBP2-19051
Formaldehyde solution 4%, buffered, pH 6.9	Millipore Sigma	1004960700
Glycerol	Sigma-Aldrich	56-81-5
ImageJ	Fiji	N/A
IntestiCult Organoid Growth Medium (Human)	STEMCELL Technologies	06010
Leica SP8 Confocal Microscope	Leica Biosystems
Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography	Millipore Sigma	L3012-10MG
Microscope Slides	Fisher Scientific	12-544-7
Normal Donkey Serum	Sigma-Aldrich	566460
Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate	Thermo Scientific	136101
PBS (Phosphate Buffered Saline), 1x [-] calcium, magnesium, pH 7.4	Corning	21-040-CM
Prolong Glass Antifade Mountant with NucBlue	Fisher Scientific	P36983
Recombinant Anti-Villin antibody [SP145]	Abcam	ab130751
Recombinant Human TNF-α protein 100 µg	Bio-Techne	210-TA-100/CF
SpectraMax iD3 Multi-Mode Microplate Reader	Molecular Devices
Thermo Forma Series II Water-Jacketed Tri-Gas Incubator, 184L	Fisher Scientific	3140
TO-PRO-3 Iodide (642/661)	Thermo Scientific	T3605
Triton X-100	Sigma-Aldrich	9002-93-1
Tubes, 0.5 mL, flat cap	Thermo Scientific	AB0350
Tween-20	Sigma-Aldrich	9005-64-5