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&#39;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.

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

<ol>
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The authors declare no conflicts of interest related to this manuscript.

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)36. Additionally, the apical epithelial surface is easily accessible for exposure to experimental stimuli, such as patient-derived microbiota or novel therapeutics. Finally, this model could also be used to study a variety of inflammatory and non-inflammatory intestinal diseases, as well as mechanisms of normal intestinal epithelial development, by utilizing enteroids derived from the relevant patient populations. This model allows for the maximization of data acquisition from a single patient sample, which is essential given the limited availability and size of neonatal intestinal samples.
Using this model, many of the physiologic changes in the intestinal epithelium that occur during NEC in infants were observed. The addition of the dysbiotic microbiome from a patient with severe NEC led to the upregulation of inflammatory cytokines (Figure 3), increased gut-on-a-chip barrier permeability, enhanced cell death, decreased proliferation, and the loss of mature epithelial sub-types36. Thus, future studies utilizing this model can focus on determining the mechanisms underlying the development of NEC and possible therapeutic interventions.
There are inherent limitations in implementing a complex model such as NEC-on-a-chip. This system requires specialized equipment, reagents, and training to utilize the microfluidics platform. In addition, the model requires close attention to detail with accurate preparation of the various media, proper seeding of the chip, and maintenance of sterile technique, being critical for success. Investigators must also have access to intestinal samples or enteroids from neonatal patients, which are generally only available at quaternary care centers with pediatric surgeons unless obtained through collaborators. Lastly, incorporation of additional important components in the pathophysiology of NEC, such as immune cells or hypoxia, further increases the difficulty of this model although increases the physiologic relevance.
In summary, NEC-on-a-chip is an important advance in the field of NEC research that will improve the quality of mechanistic studies and facilitate the testing of novel therapeutics for NEC. The ultimate goal of this research is to design and test targeted therapies that improve outcomes for infants with intestinal inflammation and NEC.

Necrotizing enterocolitis (NEC) affects preterm infants, with an incidence of up to 10% in those born weighing &#60; 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 is the limited availability and small size of primary intestinal tissue isolated from human infants. Intestinal tissue resected from infants with NEC is often necrotic and severely damaged, which complicates studies into mechanisms that precede disease onset. For example, the small intestine of infants with NEC is inundated with immune cells, and a reduced number of intestinal stem cells, decreased epithelial cell proliferation, and increased epithelial cell apoptosis are also observed4,5,6,7. This leads to difficulties in culturing intestinal epithelial cells from these samples and in isolating RNA and proteins, which can be degraded in this hostile inflammatory environment. Additionally, since the disease process is already advanced in infants with surgical NEC, mechanistic studies into factors that induce disease are unfeasible. These limitations have led to a reliance on animal models for mechanistic studies of NEC.
Animal models of NEC have been established for mice, rats, piglets, rabbits, and baboons5,8,9,11. A strength of animal&#160;models is that NEC-like intestinal disease is induced by factors associated with NEC onset in humans, including a dysbiotic microbiome, repeated episodes of hypoxia, and the absence of breast milk feeds5,8,10,11. In addition, the inflammatory response and pathologic changes observed during experimental NEC parallel human disease5,9,12. While these models mimic many of the features of human NEC, there are inherent differences between the pathophysiology of NEC in animals and humans. For example, the murine model of NEC is induced in mice born full-term, and although their intestinal development is incomplete, the pathophysiology of NEC is inherently different in this clinical context. Murine intestinal gene expression at birth is similar to a pre-viable human fetus and does not approximate that of a preterm neonate of 22-24 weeks gestation until day 14 (P14)13. This confounds the murine NEC model because intestinal injury cannot generally be induced in mice after P10. In addition, inbred strains of mice lack the immunologic14 and microbiologic diversity of human neonates15, which serves as another confounding factor. Thus, increased incorporation of primary human samples into NEC research improves the clinical relevance of studies in this field.
Studies into the mechanisms of NEC in vitro have traditionally utilized monotypic cell lines derived from adult intestinal cancer cells, such as colorectal adenocarcinoma (Caco2) and human colon adenocarcinoma (HT-29) cells16. These models are convenient but limited in physiologic relevance due to their growth from adult cancer cells, non-polarized architecture, and phenotypic changes related to repeated passages in culture. Intestinal enteroids improve upon those models since they can be grown from the crypts of intestinal tissue, differentiated into all intestinal epithelial subtypes, and form a three-dimensional (3D) villus-like structure17,18,19,20. Recently, intestinal enteroids have been combined with microfluidic technology to develop a small intestine-on-a-chip model and provide a more physiologically relevant in vitro model system21.
The initial organ-on-a-chip microfluidic devices were introduced in the early 2000s22,23,24. The first organ-on-a-chip model was the human breathing lung-on-a-chip25. This was followed by numerous single-organ models such as intestine21, liver26, kidneys27, bone marrow28, blood-brain barrier29, and heart30. These organ-on-a-chip models have been used to study acute, chronic, and rare diseases, including acute radiation syndrome,31 chronic obstructive pulmonary disease,32 and neurodegenerative diseases33. The polarized nature of the cells on these chips and the presence of two cellular compartments separated by a porous membrane allows for the modeling of complex physiologic processes such as perfusion, chemical concentration gradients, and immune cell chemotaxis34,35. These microfluidic systems thus provide a new tool for studying the pathophysiology and mechanisms of human disease.
The small intestine-on-a-chip model was described by Kasendra et al. in 2018, who utilized pediatric (ages 10-14 years old) small intestinal biopsy specimens differentiated into enteroids and cultured on a microfluidic device21. Vascular endothelial cells, continuous media flow, and stretch/relaxation were also incorporated into this model. They observed intestinal epithelial subtype differentiation, formation of 3D villus-like axes, mucus production, and small intestinal gene expression patterns21. This microfluidic model was applied to neonatal disease with the development of the NEC-on-a-chip system, which incorporates neonatal intestinal enteroids, endothelial cells, and the microbiome from a neonate with NEC36. NEC-on-a-chip recapitulates many of the critical features of human NEC, including inflammatory gene expression, loss of specialized epithelial cells, and reduced gut barrier function36. Thus, this model has numerous applications in the study of NEC, including mechanistic studies and drug discovery. In this manuscript, a detailed protocol for the performance of the NEC-on-a-chip model is provided.

<table><tbody><tr><td>[Leu15]-Gastrin I human</td><td>Sigma-Aldrich</td><td>G9145</td><td></td></tr><tr><td>A 83-01</td><td>Sigma-Aldrich</td><td>SML0788</td><td></td></tr><tr><td>Advanced Dulbecco&#039;s Modified Eagle Medium/Ham&#039;s F-12</td><td>Gibco</td><td>12634010</td><td></td></tr><tr><td>B-27 Supplement, serum free (50x)</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Basic Bio-kit</td><td>Emulate</td><td>N/A</td><td></td></tr><tr><td>BioTek Synergy 2 Multi-Mode Microplate Reader</td><td>Agilent</td><td>&amp;nbsp;7131000</td><td></td></tr><tr><td>BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro</td><td>BrandTech</td><td>759085D</td><td></td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354270</td><td></td></tr><tr><td>CFX Opus Real-Time PCR Systems</td><td>Bio-Rad</td><td>12011319</td><td></td></tr><tr><td>Chip Cradle</td><td>Emulate</td><td>N/A</td><td></td></tr><tr><td>Chip-S1 Stretchable Chip</td><td>Emulate</td><td>N/A</td><td></td></tr><tr><td>CHIR99021</td><td>Sigma-Aldrich</td><td>SML1046</td><td></td></tr><tr><td>Clear TC-treated Multiple Well Plates,&amp;nbsp; 48 well&amp;nbsp;</td><td>Corning</td><td>3548</td><td></td></tr><tr><td>Collagen from human placenta</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Collagenase, Type I, powder</td><td>Gibco</td><td>17018029</td><td></td></tr><tr><td>Complete Human Endothelial Cell Medium with Kit&amp;nbsp;</td><td>Cell Biologics</td><td>H-1168</td><td></td></tr><tr><td>Conical Polypropylene Centrifuge Tubes, 15 mL</td><td>Fisher Scientific</td><td>05-539-12</td><td></td></tr><tr><td>Conical Polypropylene Centrifuge Tubes, 50mL</td><td>Fisher Scientific</td><td>05-539-8</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Invitrogen&amp;nbsp;</td><td>C10283</td><td></td></tr><tr><td>Countess II automated cell counter</td><td>Invitrogen&amp;nbsp;</td><td>AMQAX1000</td><td></td></tr><tr><td>DAPT</td><td>Sigma-Aldrich</td><td>D5942</td><td></td></tr><tr><td>Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable</td><td>Invitrogen</td><td>&amp;nbsp;D7132</td><td>Permeability dye&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Disposable PES Filter Units, 0.2um aPES membrane</td><td>Fisher Scientific</td><td>FB12566504</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Modified Eagle&amp;prime;s Medium - high glucose</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline (DPBS)</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>EDTA, 0.5 M,&amp;nbsp; pH 8.0</td><td>Corning</td><td>46-034-CI</td><td></td></tr><tr><td>ER-1 surface activation reagent</td><td>Emulate</td><td>ER-1</td><td>Chip Activation Reagent 1</td></tr><tr><td>ER-2 surface activation reagent&amp;nbsp;</td><td>Emulate</td><td>ER-2</td><td>Chip Activation Reagent 2</td></tr><tr><td>Fibronectin Human Protein, Plasma</td><td>Gibco</td><td>33016015</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Petri Dishes with Clear Lid, 100mm</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>Gelatin-Based Coating Solution&amp;nbsp;</td><td>Cell Biologics</td><td>6950</td><td></td></tr><tr><td>Genie Temp-Shaker 300</td><td>Scientific Industries, Inc.</td><td>SI-G300</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Gibco</td><td>15750060</td><td></td></tr><tr><td>HEPES, Liquid 1M Solution (238.3 mg/ mL)</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate&amp;nbsp;</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Human Collagen Type I</td><td>Sigma-Aldrich</td><td>CC050</td><td></td></tr><tr><td>Human Primary Small Intestinal Microvascular Endothelial Cells</td><td>Cell Biologics</td><td>H-6054</td><td></td></tr><tr><td>Inverted Microscope</td><td>Fisher Scientific</td><td>03-000-013</td><td></td></tr><tr><td>Isotemp General Purpose Deluxe Water Baths</td><td>Fisher Scientific</td><td>FSGPD10</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Luria Broth (LB) agar, Miller</td><td>Supelco</td><td>L3027</td><td></td></tr><tr><td>L-WRN Cells&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-3276</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free&amp;nbsp;</td><td>Corning</td><td>356231</td><td>Cell Culture Matrix</td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>1009005</td><td></td></tr><tr><td>NAILSTAR UV LAMP</td><td>NailStar</td><td>NS-01-US</td><td></td></tr><tr><td>NanoDrop OneC&amp;nbsp;Microvolume UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td>840-274200</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>72340</td><td></td></tr><tr><td>Orb-HM1 Hub Module</td><td>Emulate</td><td>N/A</td><td></td></tr><tr><td>Paraformaldehyde</td><td>ThermoFisher</td><td>047392.9L</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Pipet-Lite Multi Pipette L8-200XLS+</td><td>Rainin</td><td>17013805</td><td></td></tr><tr><td>Pipette Tips TR LTS 1000&amp;micro;L S 768A/8</td><td>Rainin</td><td>17014966</td><td></td></tr><tr><td>Pod Portable Module</td><td>Emulate</td><td>N/A</td><td></td></tr><tr><td>Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated)&amp;nbsp;</td><td>Avantor Seradigm</td><td>1500-500</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit&amp;nbsp;</td><td>QIAGEN</td><td>205313</td><td></td></tr><tr><td>Recombinant Murine Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>315-09</td><td></td></tr><tr><td>SB 431542</td><td>Tocris</td><td>1614</td><td></td></tr><tr><td>Square BioAssay Dish with Handles, not TC-treated&amp;nbsp;</td><td>Corning</td><td>431111</td><td></td></tr><tr><td>SsoAdvanced Universal SYBR&amp;nbsp;Green Supermix</td><td>Bio-Rad</td><td>1725271</td><td></td></tr><tr><td>Steriflip-GV Sterile Centrifuge Tube Top Filter Unit</td><td>Millipore</td><td>SE1M179M6</td><td></td></tr><tr><td>Sterile Cell Strainers, 70um</td><td>Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Sterile Syringes, 10mL</td><td>Fisher Scientific</td><td>14-955-453</td><td></td></tr><tr><td>Straight, fine, sharp point scissors</td><td>Miltex&amp;nbsp;Instruments</td><td>MH5-300</td><td></td></tr><tr><td>Thermo Scientific Sorvall X4R Pro-MD Centrifuge</td><td>Thermo Scientific</td><td>75016052</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td>Detergent</td></tr><tr><td>TRIzol Reagent&amp;nbsp;</td><td>Invitrogen</td><td>15596026</td><td>RNA extraction reagent</td></tr><tr><td>Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 &amp;plusmn; 0.5</td><td>Corning</td><td>25-900-CI</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X), no phenol red&amp;nbsp;</td><td>Gibco</td><td>12604013</td><td>Enzymatic Dissociation Reagent</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>VIOS 160i CO2 Incubator, 165 L</td><td>Thermo Scientific</td><td>13-998-252</td><td></td></tr><tr><td>Y-27632</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>Zo&amp;euml;-CM1 Culture Module</td><td>Emulate</td><td>N/A</td><td></td></tr></tbody></table>

microfluidic model of necrotizing enterocolitis incorporating human neonatal intestinal enteroids and a dysbiotic microbiome

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&#39;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.

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 (Figure 2). This microfluidic device seeded with an enteroid-derived neonatal intestinal epithelium and HIMECs is known as the neonatal intestine-on-a-chip model36.
The NEC-on-a-chip model was developed to recapitulate the microbial dysbiosis present during neonatal NEC. This model includes the addition of a dysbiotic microbiome from a neonate with severe NEC to the neonatal intestine-on-a-chip model. With the addition of this dysbiotic microbiome, significantly increased expression of an array of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF&#945;;&#160;Figure 3), interleukin (IL)-1 beta (IL-1&#946;)36, and&#160;IL-8&#160;was detected36. This increase in proinflammatory cytokines mirrors what is observed for human NEC36.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65605/65605fig01.jpg" /> 
Figure 1: Schematic representation of enteroid culture and the microfluidics platform. Crypts are isolated from a surgically resected piece of neonatal intestine, and they are seeded in cell culture matrix hydrogel and grown into intestinal enteroids. Enteroids are dissociated and seeded in the top channel of the microfluidics device coated with extracellular matrix (ECM). Endothelial cells (HIMECs) are then added to the bottom channel.&#160;Figure created&#160;with Biorender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65605/65605fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65605/65605fig02.jpg" /> 
Figure 2: Progression of intestinal epithelial cells grown using the neonatal intestine-on-a-chip model. Images acquired using brightfield microscopy demonstrate neonatal enteroids on day 0, an epithelial cell monolayer in the chip on day 3, and visible villus-like structures on day 7. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65605/65605fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65605/65605fig03.jpg" /> 
Figure 3. NEC-on-a-chip intestinal epithelial cell&#160;TNF&#945;&#160;mRNA expression. Comparison of&#160;TNF&#945;&#160;mRNA levels upon incubation with media alone (control) or a dysbiotic microbiome from an infant with NEC (NEC-on-a-chip) for 24 h or 72 h. ****&#160;p&#160;&#60; 0.0001 vs. control 24 h; **&#160;p&#160;&#60; 0.005 vs. control 72 h by Mann-Whitney&#160;U&#160;test. n=9 for control 24 h; n=10 for NEC-on-a-chip 24 h; n=6 for control 72 h; n=5 for NEC-on-a-chip 72 h. Data is mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65605/65605fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Media composition for crypt isolation and enteroid growth. Refer to Van Dussen et al.40&#160;and Miyoshi et al.41&#160;for detailed methods describing the preparation of L-WRN conditioned media. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65605/table1 revised.zip" target="_blank">Please click here to download this Table.</a>
Table 2. Media composition for the NEC-on-a-chip model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65605/Frazer et al Jove Table 2 RE.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model at JoVE.com

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

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.

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

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

microfluidic-model-necrotizing-enterocolitis-incorporating-human

Nanyang Technological University

Research

JoVE Journal

Medicine

1.1K Views.  University of North Carolina at Chapel Hill. 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. 

Video: Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

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.

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.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

Immunology and Infection

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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-&#945;) 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-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, 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.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model

Source: Ares, G. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59194">A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.</a> J. Vis. Exp.(2019).
This video demonstrates the development of necrotizing enterocolitis disease in a human-derived neonatal enteroid model induced by lipopolysaccharides treatment. This model helps to study intestinal pathophysiology.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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

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A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model