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Are In Vitro Chip Models the Future of Necrotizing Enterocolitis Research?

Authors: Ioannis A. Ziogas, MD, MPH, Ankush Gosain, MD, PhD Department of Pediatric Surgery, Children's Hospital of Colorado, Aurora, CO, 80045, USA

Article Review

Lanik WE, Luke CJ, Nolan LS, et al. Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip. JCI Insight. 2023;8(8):e146496.

Necrotizing enterocolitis (NEC) is a severe, potentially fatal disease seen in premature neonates that results in intestinal injury and necrosis.1 The pathophysiology of NEC is based on loss of intestinal barrier integrity, translocation of bacteria across the gut barrier, and sepsis. The use of in vitro models is important to accelerate NEC research, given the shortage of surgically obtained samples from preterm neonates. The main limitation of currently available monotypic epithelial cell line in vitro models is the inaccurate simulation of the multiple cell types and complex intestinal dysbiosis implicated in NEC.2 On the other hand, the available intestinal organoid models can differentiate subtypes of intestinal epithelial cell and have apical-basolateral polarity within a three-dimensional spherical architecture. The limitation is the inability to assess the effect of microbial interactions or therapeutics on the apical epithelial surface.3 Notably, advances in microfluidic technology have led to the development of intestine-on-a-chip models that can simulate the human small intestine microenvironment through cellular differentiation, formation of three-dimensional villus-like axes, mucus production, continuous luminal flow, and mimicry of peristalsis.4

The article by Lanik et al.5 describes the development of a novel NEC-on-a-Chip model by a combination of premature infant small intestinal enteroids cocultured with human intestinal

microvascular endothelial cells and patient-derived microbiota. This recreates critical features of premature gut pathophysiology, including microbial dysbiosis, to simulate the pathophysiology of the human disease NEC. The authors initially demonstrated the feasibility of creating a Neonatal-Intestine-on-a-Chip model using enteroids derived from neonatal small intestine surgically resected for noninflammatory intestinal conditions that were dissociated and seeded into the Matrigel-coated microfluidic device. They showed that the Neonatal-Intestine-on-a-Chip model was similar to human small intestine by demonstrating the similarity of villus-like architecture, cell-cell adhesions, and epithelial cell apical-basolateral polarity using immunostaining.

Additionally, the authors were able to show that the gene expression in human NEC disease is associated with decreased number of stem cell, Paneth cell, and goblet cell markers, upregulation of interleukin 1β and interleukin 8, as well as reduction in the expression of proliferation markers Ki67 and proliferating cell nuclear antigen. Subsequently, they isolated the intestinal microbiome from the intestine of a premature infant that died from severe NEC and added it to their Neonatal-Intestine-on-a-Chip model to generate a NEC-on-a-Chip model and were able to show similar differences when comparing NEC-on-a-Chip to either human intestine or Neonatal-Intestine-on-a-Chip controls. Additionally, the authors were able to demonstrate the similarities between NEC-on-a-Chip and human NEC by showing a decrease in number of goblet, Paneth, and enteroendocrine cells, along with a reduction in proliferation marker Ki67 for both, compared to their respective controls of Neonatal-Intestine-on-a-Chip and healthy neonatal intestinal tissue, respectively. Then, the authors reported the effect of microbial dysbiosis on epithelial barrier dysfunction and gradually increasing permeability over 72 hours following intestinal bacteria exposure via evidence of decreased expression of an epithelial tight junction marker in the NEC-on-a-Chip model compared to control chips. Last but not least, the authors showed similar gene expression profiling in NEC-on-a-Chip and human NEC disease via RNA-sequencing, that demonstrated upregulated pathways related to inflammation and cellular death when compared to controls.

The authors concluded that the in vitro NEC-on-a-Chip model is similar in many aspects to in vivo human NEC disease, it allows study of the pathophysiology of NEC, and testing possible therapeutics, and can pave the way towards personalized medicine for NEC given the ability to produce patient-specific intestinal epithelium.

Despite the significance and novelty of the article by Lanik et al.5, there are certain limitations worth mentioning. Although the authors were able to produce three-dimensional villus-like structures, they lacked elements of complexity associated with intestinal villi in vivo,such as immune cells, lymphatics, and systemic circulation. Moreover, these were models developed under aerobic conditions without the influence of anaerobic microenvironment existing in the human intestine. Finally, due to the shortage of surgically obtained human intestine samples, the derived epithelial cells need to be expanded via in vitro models for further use that may lead to changes in gene and protein expression.

In conclusion, NEC is a significant cause of premature infant morbidity and mortality, and thus efforts to further the understanding of disease pathophysiology and novel therapeutics are of outmost importance. The development of NEC-on-a-Chip models using standardized protocols has revolutionized the field of NEC research and allows for large-scale approaches, such as drug and therapy screening. Future endeavors should focus on overcoming the inherent limitations of such models by using co-culture systems trying to imitate the immunological and microbiological components of the intestine that are implicated in the development of NEC.


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