microfluidic necrotizing enterocolitis-on-a-chip model for studying nec pathophysiology

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

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.

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

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

Research in this field is challenging because the pathophysiology of necrotizing enterocolitis is complex, and this complexity is not reflected in many commonly used in vitro models. Studies using human samples are often limited by small sample volumes and the rarity of samples obtained at the time of intestinal surgery. Our goal is to determine the mechanisms that lead to the development of NEC in preterm infants and to identify new treatment strategies.The NEC-on-a-Chip system improves upon previous models by more closely reflecting the physiology of the neonatal intestine. In this model, we combine patient-derived intestinal organoids, endothelial cells, and the microbiome from a neonate who had severe NEC onto the microfluidic chip.

Watch this Scientific Journal Video about Microfluidic Necrotizing Enterocolitis-on-a-Chip Model for Studying NEC Pathophysiology at JoVE.com

Microfluidic Necrotizing Enterocolitis-on-a-Chip Model for Studying NEC Pathophysiology

To begin, place the chip and chip carrier into the chip cradle located in a sterile tissue culture dish and keep it aside in the tissue culture hood while preparing the chip activation reagent. Add one milliliter of the CR2 solution to the vial containing CR1 powder. Transfer the solution from the vial to a 15-milliliter tube wrapped in aluminum foil.Repeat this process until four rinses and four milliliters of CR2 have been flushed through the CR1 vial. For the last rinse, cap the vial and invert it to remove residual powder. Next, add six milliliters of CR2 to the 15-milliliter tube containing CR1.Mix the final 10-milliliter solution with a pipette without introducing bubbles. Using a 200 microliter pipette tip, add 20 microliters of CR1 solution to the bottom channel inlet of the chip until the solution begins to exit the bottom channel outlet. Add 50 microliters of CR1 solution through the top channel inlet until the solution begins to exit the top channel outlet.Then, place the chips under ultraviolet light for 15 minutes. After exposure, remove any CR1 solution from the top and bottom channels. Wash both channels with 100 microliters of CR2 solution.Then, wash twice with 100 microliters of DPBS. Prepare the extracellular matrix solutions for the top and bottom channels of the chip. Add 50 microliters of extracellular matrix solution to the bottom channel inlet until the drop appears on the outlet.Then, add 50 microliters to the top channel inlet until the drop appears on the inlet. Add DPBS to the chip cradle reservoir. Place the lid on the plate and incubate the chips overnight at 37 degrees Celsius and 5%carbon dioxide incubator.The next day, flush the epithelial or top channel of the chip with 100 microliters of prewarmed chip expansion media. Then, flush the endothelial or bottom channel with 100 microliters of prewarmed HIMEC media. After counting, add 10 microliters of HIMECs to the endothelial channel.If required, add organoid growth media or chip expansion media to the epithelial channel and check the seeding density of the HIMECs. Next, invert the chip into the chip cradle placed in the cell culture dish, add DPBS to the reservoir in the chip cradle, and place the chip at 37 degrees Celsius for two to three hours to allow HIMECs to attach to the chip membrane. After incubation, return the chip cradle to an upright position.Gently wash the endothelial channel with 100 microliters of warm HIMEC media and the epithelial channel with organoid growth media or chip expansion media, leaving the media in the channel. For harvesting enteroids, gently aspirate the media and wash the wells containing cell culture matrix hydrogel with 500 microliters of warm DPBS. Add 250 microliters of cold cell recovery solution to each well and scratch the bottom of each well with a fresh pipette tip to disrupt cell culture matrix hydrogel.Add the disrupted cell suspension to a cold 15-milliliter tube on ice and incubate for 45 minutes. After incubation, centrifuge the suspension and remove the supernatant. Then, add two milliliters of warm enteroid dissociation media to the pellet and place the tube in a 37 degrees Celsius water bath.For 60 to 120 seconds, next, add 10 milliliters of cold tissue washing media to the tube. Then, centrifuge and remove the supernatant before resuspending the pellet in 200 microliters of chip expansion media. Using a P200 pipette, dissociate the enteroids and add the suspension into a 1.5-milliliter micro centrifuge tube.Count the cells and adjust the volume of chip expansion media to achieve a concentration of six times 10 to the six cells per milliliter. Then, add 30 microliters of resuspended cell suspension to the top channel of the chip. After returning the chips to the chip cradle, add DPBS to the reservoir and incubate the chips overnight at 37 degrees Celsius and 5%carbon dioxide.The next day, flush the epithelial channel twice with 100 microliters of chip expansion media and the endothelial channel with 100 microliters of HIMEC media. Then, add two milliliters of the chip expansion media to the top channel inlet reservoir and 300 microliters to the top channel outlet reservoir. Add two milliliters of the HIMEC media to the bottom channel inlet reservoir and 300 microliters to the bottom channel outlet reservoir.Prime the pods and add the chips to the pods. Then add the pods to the culture module. Set a flow rate of 30 microliters per hour and initiate the cycle.The intestinal epithelial cells grown using the neonatal intestine on a chip formed a confluent cell monolayer, and subsequently developed into mature three-dimensional villus-like structure. The addition of a dysbiotic microbiome from a neonate with severe necrotizing enterocolitis to the neonatal intestine on a chip model showed increased expression of TNF-alpha compared to the control.

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162 visualizações.  University of North Carolina at Chapel Hill. Para começar, coloque o chip e o portador de chips no suporte de chips localizado em uma placa de cultura de tecido estéril e mantenha-o de lado no capuz de cultura de tecidos enquanto prepara o reagente de ativação de chip. Adicione um mililitro da solução de CR2 ao frasco para injetáveis contendo pó de CR1. Transfira a solução do frasco para injetáveis para um tubo de 15 mililitros envolto em papel alumínio.Repita este processo até que quatro enxaguamentos e quatro milil...