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A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis
In This Article
Summary
Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.
Abstract
Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.
Introduction
Human enteroids are an ex vivo 3-dimensional culture system generated from stem cells isolated from intestinal crypts of human intestinal tissue samples. This ground-breaking model was pioneered by Hans Clevers et al. in 2007 following the discovery of Lgr5+ stem cells at the crypts of small intestine in mice1. Their work laid the foundation for establishing an ex vivo intestinal epithelial culture of multiple cell types that could be passaged without significant genetic or physiologic changes2. Since this discovery, enteroids have been used as a novel model to study normal digestive physiology, and the pathophysiology o....
Protocol
Institutional review board approval was obtained (IRB #2013-15152) for collection of tissue samples from patients undergoing bowel resection at Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL. All protocols were performed in compliance with institutional and national guidelines and regulations for human welfare. Written informed parental consent was required and obtained prior to sample collection in all cases.
1. Reagent Preparation
- Prepare Culture Media st.......
Representative Results
Immediately after plating, the freshly isolated intestinal crypts appear as elongated rods. Within hours, the enteroid will take on a round appearance (Figure 1a). Over the next several days, the enteroids will start forming spheres as seen in Figure 1b. Budding should occur between 5-10 days (Figure 1c) and enteroid collection should occur at that t.......
Discussion
This novel ex vivo human intestinal enteroid model serves as a useful method for the study of intestinal barrier dysfunction in necrotizing enterocolitis (NEC). The enteroid processing methods presented here were adapted from the previous work of Drs. Misty Good, Michael Helmrath and Jason Wertheim10,11,12.
Details surrounding the whole tissue collection and timing of crypt isolation are critical step.......
Acknowledgements
This work was supported by the National Institute of Health Institute of Diabetes and Digestive and Kidney Disease Grant (K08DK106450) and the Jay Grosfeld Award from the American Pediatric Surgical Association to C.J.H.
....Materials
| Name | Company | Catalog Number | Comments |
| 4% Paraformaldehyde | ThermoFisher | AAJ19943K2 | |
| A-83 | R&D Tocris | 2939/10 | |
| Amphotericin B | ThermoFisher | 15290026 | |
| B-27 supplement minus Vitamin A | ThermoFisher | 17504-044 | |
| Basement Membrane Matrix (Matrigel) | Corning | CB-40230C | |
| DMEM/F-12 | ThermoFisher | MT-16-405-CV | |
| Dulbecco’s Modified Eagle Medium (DMEM) | ThermoFisher | 11-965-118 | |
| Dulbecco’s Phosphate-Buffered Saline (DPBS) | ThermoFisher | 14190-144 | |
| Epidermal Growth Factor (EGF) | Sigma | E9644-.2MG | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma | EDS-500G | |
| Fetal Bovine Serum (FBS) | Gemini Bio-Pro | 100-125 | |
| Gentamicin | Sigma | G5013-1G | |
| GlutaMAX (L-glutamine) | ThermoFisher | 35050-061 | |
| Insulin | Sigma | I9278-5mL | |
| [leu] 15-gastrin 1 | Sigma | G9145-.1MG | |
| Lipopolysaccharide (LPS) | Sigma | L2630-25MG | |
| N-2 supplement | ThermoFisher | 17502-048 | |
| N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) | ThermoFisher | 15630-080 | |
| N-Acetylcysteine | Sigma | A9165-5G | |
| Nicotinamide | Sigma | N0636-100G | |
| Noggin | R&D Systems INC | 6057-NG/CF | |
| Penicillin-Streptomycin | ThermoFisher | 15140-148 | |
| Phosphate Buffered Saline (PBS) | Sigma | P5368-5X10PAK | |
| RPMI 1640 Medium | Invitrogen | 11875093 | |
| R-Spondin | PEPROTECH INC | 120-38 | |
| SB202190 | Sigma | S7067-5MG | |
| Tissue Processing Gel (Histogel) | ThermoFisher | 22-110-678 | |
| Wnt3a | R&D Systems INC | 5036-WN-010 | |
| Y-27632 | Sigma | Y0503-1MG |
References
- Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
- Zachos, N. C., et al.
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