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Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Published: July 28th, 2023



1Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, 2University of Nebraska College of Medicine, 3Department of Surgery, Washington University School of Medicine

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field's understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Necrotizing enterocolitis (NEC) affects preterm infants, with an incidence of up to 10% in those born weighing < 1500 g1. The pathophysiology of NEC is complex and includes damage to the intestinal epithelium, disruption of intestinal tight junctions, increased gut barrier permeability, immune dysregulation, and epithelial cell death2,3. Our understanding of the mechanisms involved in the pathogenesis of NEC remains incomplete, and despite decades of research, there are still no effective targeted therapies.

A significant barrier to advancing NEC research....

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Enteroids were derived from small intestinal samples from premature infants (born at 22 to 36 weeks gestation) obtained at the time of surgery for NEC or other intestinal conditions with non-inflammatory etiologies. All specimen collection and processing was performed after informed consent and approval from the Institutional Review Boards at Washington University in St. Louis (IRB Protocol numbers 201706182 and 201804040) and the University of North Carolina at Chapel Hill (IRB protocol number 21-3134).

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Enteroids were seeded onto the microfluidic device (Figure 1) and cultured as described above. Growth of the enteroids in cell culture matrix hydrogel prior to seeding and then the subsequent expansion of the intestinal epithelial cell monolayer after seeding the device was monitored via brightfield microscopy (Figure 2). A confluent intestinal epithelial cell monolayer formed and subsequently developed into a mature 3D villus-like structure (

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This NEC-on-a-chip system is a powerful new tool that can be used to model the pathophysiology of NEC. This platform provides a complex microenvironment that more closely resembles the in vivo intestinal milieu than previous models by incorporating a co-culture system with continuous luminal flow and stretch. These conditions promote the development of 3D villus-like architecture lined by a highly polarized epithelium consisting of mature epithelial sub-types and tight junctions (Figure 2

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This manuscript was supported by R01DK118568 (MG), R01DK124614 (MG), and R01HD105301 (MG) from the National Institutes of Health, the Chan Zuckerberg Initiative Grant 2022-316749 (MG), a Thrasher Research Fund Early Career Award (LCF), a UNC Children's Development Early Career Investigator Grant (LCF) through the generous support of donors to the University of North Carolina at Chapel Hill, and the Department of Pediatrics at the University of North Carolina at Chapel Hill.


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Name Company Catalog Number Comments
[Leu15]-Gastrin I human Sigma-Aldrich G9145
A 83-01 Sigma-Aldrich SML0788
Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 Gibco 12634010
B-27 Supplement, serum free (50x) Gibco 17504044
Basic Bio-kit Emulate N/A
BioTek Synergy 2 Multi-Mode Microplate Reader Agilent  7131000
BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro BrandTech 759085D
Cell Recovery Solution Corning 354270
CFX Opus Real-Time PCR Systems Bio-Rad 12011319
Chip Cradle Emulate N/A
Chip-S1 Stretchable Chip Emulate N/A
CHIR99021 Sigma-Aldrich SML1046
Clear TC-treated Multiple Well Plates,  48 well  Corning 3548
Collagen from human placenta Sigma-Aldrich C5533
Collagenase, Type I, powder Gibco 17018029
Complete Human Endothelial Cell Medium with Kit  Cell Biologics H-1168
Conical Polypropylene Centrifuge Tubes, 15 mL Fisher Scientific 05-539-12
Conical Polypropylene Centrifuge Tubes, 50mL Fisher Scientific 05-539-8
Countess Cell Counting Chamber Slides Invitrogen  C10283
Countess II automated cell counter Invitrogen  AMQAX1000
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) Invitrogen D3571
DAPT Sigma-Aldrich D5942
Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable Invitrogen  D7132 Permeability dye 
Dimethyl sulfoxide (DMSO) Sigma-Aldrich D8418
Disposable PES Filter Units, 0.2um aPES membrane Fisher Scientific FB12566504
DMEM/F-12 Gibco 11320033
Donkey serum Sigma-Aldrich D9663
Dulbecco′s Modified Eagle′s Medium - high glucose Sigma-Aldrich D5796
Dulbecco′s Phosphate Buffered Saline (DPBS) Gibco 14190-136
EDTA, 0.5 M,  pH 8.0 Corning 46-034-CI
ER-1 surface activation reagent Emulate ER-1 Chip Activation Reagent 1
ER-2 surface activation reagent  Emulate ER-2 Chip Activation Reagent 2
Fibronectin Human Protein, Plasma Gibco 33016015
Fisherbrand Petri Dishes with Clear Lid, 100mm Fisher Scientific FB0875713
Gelatin-Based Coating Solution  Cell Biologics 6950
Genie Temp-Shaker 300 Scientific Industries, Inc. SI-G300
Gentamicin  Gibco 15750060
HEPES, Liquid 1M Solution (238.3 mg/ mL) Corning 25-060-CI
Hoechst 33342, Trihydrochloride, Trihydrate  Invitrogen H3570
Human Collagen Type I Sigma-Aldrich CC050
Human Primary Small Intestinal Microvascular Endothelial Cells Cell Biologics H-6054
Inverted Microscope Fisher Scientific 03-000-013
Isotemp General Purpose Deluxe Water Baths Fisher Scientific FSGPD10
L-Glutamine  Gibco 25030-081
Luria Broth (LB) agar, Miller Supelco L3027
L-WRN Cells  American Type Culture Collection CRL-3276
Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free  Corning 356231 Cell Culture Matrix
N-2 Supplement (100x) Gibco 17502048
N-acetyl-L-cysteine Sigma-Aldrich 1009005
NanoDrop OneC Microvolume UV-Vis Spectrophotometer Thermo Scientific 840-274200
Nicotinamide Sigma-Aldrich 72340
Orb-HM1 Hub Module Emulate N/A
Paraformaldehyde ThermoFisher 047392.9L
Penicillin-Streptomycin  Gibco 15140122
Phosphate buffered saline (PBS) Gibco 10010023
Pipet-Lite Multi Pipette L8-200XLS+ Rainin 17013805
Pipette Tips TR LTS 1000µL S 768A/8 Rainin 17014966
Pod Portable Module Emulate N/A
Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated)  Avantor Seradigm 1500-500
QuantiTect Reverse Transcription Kit  QIAGEN 205313
Recombinant Murine Epidermal Growth Factor (EGF) PeproTech 315-09
SB 431542 Tocris 1614
Square BioAssay Dish with Handles, not TC-treated  Corning 431111
SsoAdvanced Universal SYBR Green Supermix Bio-Rad 1725271
Steriflip-GV Sterile Centrifuge Tube Top Filter Unit Millipore SE1M179M6
Sterile Cell Strainers, 70um Fisher Scientific 22-363-548
Sterile Syringes, 10mL Fisher Scientific 14-955-453
Straight, fine, sharp point scissors Miltex Instruments MH5-300
Thermo Scientific Sorvall X4R Pro-MD Centrifuge Thermo Scientific 75016052
Triton X-100  Sigma-Aldrich T8787 Detergent
TRIzol Reagent  Invitrogen 15596026 RNA extraction reagent
Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 ± 0.5 Corning 25-900-CI
TrypLE Express Enzyme (1X), no phenol red  Gibco 12604013 Enzymatic Dissociation Reagent
Trypsin-EDTA solution Sigma-Aldrich T4174
VIOS 160i CO2 Incubator, 165 L Thermo Scientific 13-998-252
Y-27632 Tocris 1254
Zoë-CM1 Culture Module Emulate N/A

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